Glycosylated analogs of camptothecin

ABSTRACT

The present invention relates to chemotherapeutic agents, and more particularly, to novel analogs of camptothecin. The camptothecin analogs display increased solubility through the hydrophilicity of added non-ionic sugar substituents. In accordance with the present invention, a member from the class of novel camptothecin analogs is to be delivered in vivo as a chemotherapeutic agent to fight cancer growth in the body.

FIELD OF THE INVENTION

The present invention relates to chemotherapeutic agents, and moreparticularly, to novel analogs of camptothecin.

BACKGROUND OF THE INVENTION

Despite the enormous efforts and resources directed at finding a cure,cancer remains an elusive and deadly foe for mankind. The standardmethods of treatment usually include chemotherapy, radiation treatment,and surgical removal or tumors and/or growths, or some combinationthereof. These treatments, combined with an emphasis on preventativelifestyle modification, have afforded a measure of success in the battleagainst some cancers. However, cancer remains one of the leading causesof mortality, and cancers detected at matured stages are invariablyfatal.

Numerous chemical agents have been devised for the treatment of cancerwith varying degrees of efficacy. However, no single drug has onehundred percent effectiveness against different cancers, and negativeside-effects ranging from minor to serious are always present.

Recently, there has been much research directed toward the use ofcamptothecin and its derivatives to fight cancer. Isolated in 1966 fromthe Chinese tree Camptotheca acuminata, camptothecin was found to havesignificant efficacy in animal tumor models. Upon advancement to humanclinical studies, camptothecin was found to have mixed results infighting tumor growth and possessed side effects ranging from vomitingand diarrhea to myelosuppression and hemorrhagic cystitis. The sideeffects were so severe that Phase II clinical trials were eventuallydiscontinued in the United States.

It is believed that camptothecin has a unique mechanism of action, i.e.,via topoisomerase I DNA damage by binding and stabilizing a covalentDNA-topoisomerase I complex in which one of the DNA strands is broken.See Slichenmyer et al., "The Current Status Of Camptothecin Analogues AsAntitumor Agents," J. Nat'l. Cancer Inst. 85:2 (1993) and referencescited therein!. Among the formidable challenges facing any effort todevelop the potential anticancer properties of camptothecin into auseable treatment are clearly the problems of drug delivery andtoxicity.

Drug delivery is complicated by the fact that camptothecin iswater-insoluble in its unmodified state. To this end several derivativesof camptothecin have been developed in order to address those twoproblems. In 1991, Kingsbury et al. see "Synthesis Of Water-Soluble(Aminoalkyl)camptothecin Analogues: Inhibition Of Topoisomerase I AndAntitumor Activity," J. Med. Chem. 34:98(1991)! described the synthesisof several water-soluble analogs of camptothecin, by introduction ofaminoalkyl groups into the camptothecin ring system. One of theseanalogs in particular, (s)-9-dimethylaminomethyl-10-hydroxycamptothecinhydrochloride!, later referred to as topotecan or TPT, was found to havesignificant anti-tumor effects when tested against various carcinomacells in an in vitro clongenic assay. In animal studies, TPT was foundto be at least as good or better than camptothecin in its effectivenessagainst tumor growth for a variety of cancers.

In human clinical studies, TPT was found to be clearly more promisingthan camptothecin in its efficacy. However, the derivative still causedseveral significant side effects, including myelosuppression,neutropenia, and thrombocytopenia.

Sawada et al. see "Synthesis And Antitumor Activity Of20(S)-Camptothecin Derivatives: Carbamate-Linked, Water SolubleDerivatives Of 7-Ethyl-10-hydroxycamptothecin," Chem. Pharm. Bull39:1446 (1991)! prepared several derivatives of camptothecin by bondingthe phenolic hydroxyl group of 7-ethyl-10-hydroxycamptothecin withdiamines through a monocarbamate linkage. These diamine derivatives weremade water-soluble by conversion to their hydrochloride salts. Onederivative in particular, 7-ethyl-10-4-(piperidino)-1-piperidino!carboxylcamptothecin hydrochloride("CPT-11"), possessed significant in vivo preclinical activity; itexhibited negligible in vitro activity as shown by its inability toinhibit Topoisomerase I activity see Kingsbury et al. supra! and tumorcell growth see Kingsbury et al. supra and Kawato et al., "IntracellularRoles Of SN-38, A Metabolite Of The Camptothecin Derivative CPT-11, InThe Antitumor Effect Of CPT-11," Cancer Res. 51:4187 (1991)!, due to thefact that CPT-11 must first be metabolized by the body into itsbioreactive form SN-38; thus making CPT-11 a "pro-drug." In fact, the invivo preclinical anti-cancer effects of CPT-11 were more significant andwider ranging than either camptothecin or any other analog synthesizedto date.

However, the promising effects of CPT-11 were still accompanied bysignificant side effects when the studies progressed to human clinicaltrials. Although the broad range of side effects was not generallypresent for CPT-11, severe respiratory complications combined withconstant diarrhea pose significant challenges for CPT-11 basedchemotherapy. See Slichenmeyer et al. supra!.

Clearly, there remains a need for a powerful anti-cancer derivative ofcamptothecin which does not possess significant side effects in humansundergoing treatment with such derivatives.

SUMMARY OF THE INVENTION

The present invention relates to chemotherapeutic agents, and moreparticularly, to novel analogs of camptothecin. A camptothecin"derivative" or "analog" of the present invention has the fundamentalstructure of unmodified camptothecin (see FIG. 1). The contemplatedcamptothecin analogs possess the substructure of unmodified camptothecinsubstituted with either one or two side chains or one optionallysubstituted, fused, carbocyclic or heterocyclic ring and one side chainwherein the side chains or ring substituents are independently selectedfrom: lower alkyl, lower alkenyl, lower alkoxy, lower alkylamino,di-lower alkylamino, lower alkoxy-lower alkyl, lower alkylamino-loweralkyl, or dilower alkylamino-lower alkyl; and where one or two hydroxylsare attached at any of the methyl or methylene carbons of these appendedchains or rings; and wherein further at least one of these primary orsecondary alcohols is glycosylated with a saccharide substituent whichmay be exemplified by but is not limited to2,3-desoxy-2,3-dehydroglucose, 2,3-desoxy-2,3-dehydroglucose diacetate,glucoside, glucoside tetraacetate, mannoside, mannoside tetraacetate,galactoside, galactoside tetraacetate, alloside, alloside tetraacetate,guloside, guloside tetraacetate, idoside, idoside tetraacetate,taloside, taloside tetraacetate, rhamnoside, rhamnoside triacetate,maltoside, maltoside heptaacetate, 2,3-desoxy-2,3-dehydromaltoside,2,3-desoxy-2,3-dehydromaltoside pentaacetate, 2,3-desoxymaltoside,lactoside, lactoside tetraacetate, 2,3-desoxy-2,3-dehydrolactoside,2,3-desoxy-2,3-dehydrolactoside pentaacetate, 2,3-desoxylactoside,glucouronate, N-acetylglucosamine. In one embodiment, the presentinvention contemplates a camptothecin analog modified at the sevenposition by chemical, enzymatic, or biological means, such that itcontains carbohydrate moieties. (See e.g., FIGS. 4-15).

The embodiments of the present invention contemplate the camptothecinbasic skeleton glycosylated by a wide variety of sugar moieties. Thesugar moieties are connected to the basic camptothecin skeleton bylinker side chains or rings containing primary or secondary alcohols.Camptothecin having such linker side-chains or rings (in preparation forthe addition of sugar moieties) are referred to as "modifiedcamptothecins". The molecular architecture unites the camptothecinsubstructure, which carries the anti-cancer activity, with the sugarsubstructure, which confers the hydrophilic-lipophilic balance. Thecombination is achieved to allow both sufficient durability to enabledrug transport and delivery and sufficient flexibility so that thecamptothecin subgroup may (if necessary) be disconnected by enzymaticand/or hydrolytic mechanisms. While not intending to limit the inventionto any particular mechanism, it is believed that the linker and thesugar together perturb the crystal lattice of the camptothecin analog soas to remove the high crystal binding forces which make manycamptothecin-like substances highly insoluble not just in water but inpractically all solvents.

The attempted solubilization of camptothecin-like molecules is not new.In the prior art however this solubilization is achieved principallythrough the use of salts of basic amino groups which are attached eitherdirectly or indirectly to the camptothecin skeleton. In contrast, thepresent invention contemplates increasing solubilization through thehydrophilicity of non-ionic sugar substituents. Although a minority ofstructures which fall within the scope of this invention do containbasic nitrogens these are either part of the sugar or simply constitutea part of the linker unit.

Camptothecin glycosides have been previously described, for example oneis a natural product called chaboside see Tetrahedron Lett. 31:5169(1990) and "10-Hydroxycamptothecin Glycosides as Antitumor Agents" JP63238098! The glycosides heretofore described, however, have the sugarmoiety directly bonded through a simple oxygen to the camptothecinsubstructure (FIG. 1 ). These phenolic glycosidic links are readilyhydrolyzable. Consequently these prior art molecules lack the durabilityof the analogs of the present invention.

A wide variety of sugar units indirectly linked to the camptothecinsubstructure are contemplated. The modes of preparation of alcohol-sugarglycoside linkages are well known to practitioners of the art as isevidenced by the comprehensive reviews addressing this particular typeof bond formation see K. Toshima and K. Tatsuta, "Recent progress inO-glycosylation methods and its application to natural productssynthesis" Chem. Rev. 1503 (1993); R. R. Schmidt "New methods for thesynthesis of glycosides and oligosaccharides-Are there alternatives tothe Koenigs-Knorr method?" Angew. Chem. Int. Ed. Engl. 25:212 (1986);and N. K. Kochetkov "Recent developments in the synthesis ofpolysaccharides and stereospecificity of the glycosylation reactions,"Stud. Nat. Prod. Chem. 14:201 (1994)!. The glycosidic bonds required inthe present invention are prepared using these glycosylation techniqueseither on the modified camptothecin itself or on the more soluble C-20hexanoate whose synthesis is described herein in the experimentalsection. The hexanoate solubilizing group may be easily removed, forexample during the deprotection of the sugar protecting groups. Thepresent invention contemplates molecules containing the camptothecinsubstructure which when glycosylated produce the camptothecin glycosideanalog of the present invention. The corresponding aglycons are eitheralready described in the chemical literature or are easily synthesizedby standard reactions from substances whose preparation is alreadydescribed in the chemical literature. Examples of previously describedcompounds which fall within the scope of the present invention whenglycosylated include 5-hydroxymethylcamptothecins;5,5-Bis(hydroxymethyl)camptothecin; 7-N,N-(2-hydroxyethyl)aminomethyl!-10,11-ethylenedioxycamptothecin; 7-N-methyl-N-(2-hydroxyethyl)aminomethyl!-10,11-ethylenedioxycamptothecin;7-N-methyl-N-(2-hydroxyethyl)aminomethyl!-10,11-methylenedioxycamptothecin.Examples of previously described compounds which are readily convertedinto new camptothecin analogs include 7-chlorocamptothecin,9-bromocamptothecin, 9-chlorocamptothecin, 10-bromocamptothecin,10-chlorocamptothecin, 11-bromocamptothecin, 11-chlorocamptothecin,12-bromocamptothecin, and 12-chlorocamptothecin. Other compounds withinthe scope of this invention which are also contemplated are10-(2-hydroxyethoxy)camptothecin, and 10-2,3-dihydroxy-propoxy!camptothecin.

In one embodiment, the present invention contemplates an analog that isglycosylated on a 7-position side-chain. One embodiment of the presentinvention contemplates a camptothecin that is substituted at the 7position by a lower alkyl group functionalized at a primary or secondarycarbon by a hydroxyl through which is connected a sugar. All may beoptionally substituted in the 10,11 positions with either amethylenedioxy or ethylenedioxy ring. One embodiment of the presentinvention contemplates glycosylation of the modified camptothecin,7-hydroxymethylcamptothecin. The synthesis of the corresponding aglyconsmay be facilitated by using the C-20 hexanoate of camptothecin as analternate substrate or by manipulation of the opened lactone formfollowed by subsequent reclosure. In another embodiment, the presentinvention contemplates an analog having a linker at the 7 position thatallows for glycosylation. While not limited to particular linkers, apreferred linker is oxymethyl. One analog of the present invention is 7-4,6-di-O-acetyl-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin. Another analog of the present invention is 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin. Another analog of the present invention is 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin.Another analog of the present invention is 7-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl camptothecin. Another analogof the present invention is 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin.Another analog of the present invention is 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin.Another analog of the present invention is 7-4,6-di-O-acetyl-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.Another analog of the present invention is 7- 2,3dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin. Anotheranalog of the present invention is 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.Anther analog of the present invention is 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.Another analog of the present invention is 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.Yet another analog of the present invention is 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.

In one embodiment, an analog of the present invention is synthesized bya) providing in any order: i) unmodified camptothecin, ii) a modifyingreagent, iii) a derivatizing reagent, and iv) a catalyst; b) reacting inany order: i) unmodified camptothecin and ii) a modifying reagent toform a modified camptothecin; c) reacting in any order: i) the modifiedcamptothecin of step (b), ii) a derivatizing reagent, and iii) acatalyst to form a chemotherapeutic anti-cancer analog of thecamptothecin molecule of FIG. 1.

Modifying reagents are those reagents which will act on unmodifiedcamptothecin to add a side-chain, for example oxymethyl. In oneembodiment the modifying reagent is an alcohol (e.g. methanol).Derivatizing reagents are those reagents which provide the substituentsadded to modified camptothecin. In one embodiment, the derivatizingreagent is a carbohydrate glycal. In one embodiment the carbohydrateglycal is selected from the group consisting of glucose glycal (glucal),maltose glycal (maltal), and lactose glycal (lactal). A catalyst, ingeneral, is a substance which increases the rate of a chemical reaction.In this particular reaction, a catalyst is a substance which increasesthe rate of formation of the glycosylated camptothecin analog. In oneembodiment, the catalyst is a molecular diatomic halogen. In oneembodiment, the molecular diatomic halogen is molecular diatomic iodine.

In one embodiment, the carbohydrate glycal is a disaccharide glycal, forexample maltose glycal (maltal), and is synthesized by a) providing inany order: i) unmodified disaccharide, ii) a protecting reagent, iii) adisaccharide derivatizing reagent, and iv) a reducing agent; b) reactingin any order: i) unmodified disaccharide and ii) a protecting reagent toform a protected disaccharide; c) reacting in any order: i) theprotected disaccharide of step (b) and ii) a disaccharide derivatizingreagent to form a derivatized protected disaccharide; d) reacting in anyorder: i) the derivatized protected disaccharide of step (c) and ii) anreducing agent to form a disaccharide glycal. In one embodiment theunmodified disaccharide is maltose. In another embodiment the unmodifieddisaccharide is lactose. Protecting reagents are those reagents whichprotect particular functionalities of the disaccharide from beingdestroyed in subsequent reactions. In one embodiment the protectingreagent is an esterifying reagent, for example acetic anhydride.Disaccharide derivatizing agents are those reagents which convert adisaccharide into a disaccharide halide. In one embodiment, thedisaccharide derivatizing reagent is a halogenating reagent, for examplehydrobromic acid. In one embodiment, the reducing agent is Zn/CuSO₄.

In one embodiment, the activated carbohydrate glycal is an activateddisaccharide glycal. Activated disaccharide glycals are those glycalswhich have sufficient reactivity to readily react with modifiedcamptothecins to form glycosylated camptothecin analogs in high yields(as compared to yields with unactivated disaccharide glycals), forexample pentaacetylbenzoyl glycal, and are synthesized by a) providingin any order: i) disaccharide glycal, ii) an activating reagent, andiii) a catalyst; b) reacting in any order: i) the disaccharide glycal,ii) a protecting reagent, and iii) a catalyst to form an activateddisaccharide glycal. In one embodiment the disaccharide glycal ismaltal. Activating reagents are those reagents that convert disaccharideglycals into activated disaccharide glycals. In one embodiment theactivating reagent is a carboxylic acid, for example o-anisic acid. Inthis particular reaction a catalyst is a substance which increases therate of formation of the activated disaccharide glycal. In oneembodiment, the catalyst is a molecular diatomic halogen. In oneembodiment, the molecular diatomic halogen is molecular diatomic iodine.

In one embodiment, the present invention contemplates a method ofsynthesizing a chemotherapeutic anti-cancer glycosylated analog of thecamptothecin molecule of FIG. 1, comprising the steps: a) providing amodified camptothecin; b) synthesizing a disaccharide glycal; c)treating said disaccharide glycal so as to generate an activateddisaccharide glycal; and d) reacting in any order: i) said modifiedcamptothecin of step (a), ii) said activated disaccharide glycal of step(c), and iii) a catalyst, under conditions so as to form achemotherapeutic anti-cancer analog of the camptothecin molecule ofFIG. 1. In one embodiment, the modified camptothecin of step (a) isprepared by reacting unmodified camptothecin with methanol and moleculardiatomic iodine to form a modified camptothecin. In one embodiment, thedisaccharide glycal of step (b) is synthesized according to thefollowing procedure: 1) providing in any order: i) unmodifieddisaccharide, ii) acetic anhydride, iii) hydrobromic acid, and iv)zinc/cuprous sulfate; 2) reacting said unmodified disaccharide with saidacetic anhydride to form a protected disaccharide; 3) reacting saidprotected disaccharide of step (2) with said hydrobromic acid to form aderivatized protected disaccharide; and 4) reacting said derivatizedprotected disaccharide of step (3) with said zinc/cuprous sulfate toform a disaccharide glycal. In one embodiment, the treating in step (c)comprises reacting in any order: i) said disaccharide glycal, ii)o-anisic acid, and iii) molecular diatomic iodine to form an activateddisaccharide glycal. In one embodiment, said catalyst is a moleculardiatomic halogen, for example molecular diatomic iodine.

It should be clear that the order of the steps is, in some instances,variable. For example, while the synthesis of the disaccharide glycalmust precede the treating step creating the activated disaccharideglycal, the preparation of the modified camptothecin is independent ofthe creation of the activated disaccharide glycal (i.e. it may be donebefore, during, or after the synthesis of the activated disaccharideglycal) and the only temporal requirement is that the activateddisaccharide glycal and the modified camptothecin are prepared beforetheir eventual reaction (in step (d)) to form the glycosylatedcamptothecin analog of the present invention.

The analogs of the present invention have numerous uses. First, they maybe successfully employed as standards for analytical techniques (e.g.,HPLC) so that new analogs can be easily identified. In addition, thepresent invention also contemplates in vivo use; in accordance with thepresent invention, a member from the class of novel camptothecin analogsis to be delivered as a chemotherapeutic agent to fight cancer growth inthe body.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structure of unmodified camptothecin.

FIG. 2 shows the structure of (s)-9-dimethylaminomethyl-10-hydroxy-camptothecin-hydrochloride! (topotecan or TPT) a previously describedcamptothecin derivative of the prior art (A), its lactone ring openedform (B), and the structure of 7-ethyl-10-4-(piperidino)-1-piperidino!carboxylcamptothecin hydrochloride (CPT-11)another previously described camptothecin derivative of the prior art(C).

FIG. 3 shows the structure of 7-hydroxymethyl camptothecin, anintermediate in the chemical synthesis of the camptothecin analogs ofthe present invention.

FIG. 4 shows the structure of a preferred camptothecin analog of thepresent invention, 7-4,6-di-O-acetyl-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin(HAR4).

FIG. 5 shows the structure of a preferred camptothecin analog of thepresent invention, 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin(HAR5).

FIG. 6 shows the structure of a preferred camptothecin analog of thepresent invention, 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin(HAR6).

FIG. 7 shows the structure of a preferred camptothecin analog of thepresent invention, 7-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin (HAR7).

FIG. 8 shows the structure of a preferred camptothecin analog of thepresent invention, 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin.

FIG. 9 shows the structure of a preferred camptothecin analog of thepresent invention, 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin

FIG. 10 shows the structure of a preferred camptothecin analog of thepresent invention, 7-4,6-di-O-acetyl-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.

FIG. 11 shows the structure of a preferred camptothecin analog of thepresent invention, 7-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.

FIG. 12 shows the structure of a preferred camptothecin analog of thepresent invention, 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxyα-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.

FIG. 13 shows the structure of a preferred camptothecin analog of thepresent invention, 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.

FIG. 14 shows the structure of a preferred camptothecin analog of thepresent invention, 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin.

FIG. 15 shows the structure of a preferred camptothecin analog of thepresent invention, 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl-oxymethylcamptothecin.

FIG. 16 shows the structure of two prior art camptothecin analogsaltered at the 20 position.

FIG. 17 shows the structure of preferred glycosylation reaction glycalsof the formulas I-III and Ia-IIIa.

FIG. 18 shows the lack of inhibition of relaxation of supercoiled DNA bycamptothecin analogs of the present invention which possessed chemicalmodification at the 20 position.

FIG. 19 shows the inhibition of relaxation of supercoiled DNA by thepreferred camptothecin analogs of the present invention (HAR4, HAR5,HAR6, and HAR7).

FIG. 20 shows the structure of a camptothecin analog of the presentinvention, 9-ethyl-1-(4-O-α-D-glucopyranosyl-α-D-galactopyranosyl)oxy!-1,2,3,9,12,15-hexahydro-9-hydroxy-2-methyl-10H,13H-Benzo ij!pyrano 3',4':6,7!indolizino 1,2-c!2,6!naphthyridine-10,13-dione.

DESCRIPTION OF THE INVENTION

The present invention relates to chemotherapeutic agents, and moreparticularly, to novel analogs of camptothecin. The description of thepresent invention involves: (I) Camptothecin and Previously DescribedDerivatives; (II) Properties of Camptothecin Analogs of the PresentInvention; (III) Synthesis of Novel Glycosylated Camptothecin Analogs;(IV) Methodology for Screening Camptothecin Analogs; and (V) In VivoUses.

I. Camptothecin And Previously Described Derivatives

A. Physical Properties

Camptothecin in its unmodified form is quite insoluble in water (20μg/mL). As a result, the sodium salt of camptothecin, which is watersoluble, was used in clinical trials, although the anti-cancer efficacyof the sodium salt was much less than unmodified camptothecin.

The two camptothecin derivatives which had received the most interestfor their possible anti-cancer effects were CPT-11 and TPT. Bothcompounds exhibited improvement in the area of solubility, compared tocamptothecin. CPT-11, which was administrated as its hydrochloridetrihydrate had a water solubility of 32 mg/mL. TPT, which wasadministered as its hydrochloride salt had a water solubility of 1mg/mL. See Kingsbury et al. supra!.

B. Biological Properties

Camptothecin was used as a control to test the anti-cancer efficacy ofthe novel camptothecin analogs of the present invention. Two types of invitro assays were used to measure the effectiveness of both modifiedcamptothecin and the analogs of the present invention: first, thecompound of interest was utilized in the well-established topoisomeraseI assay, to determine the degree to which the drag inhibited theactivity of topoisomerase I; second, the compound of interest was testedto determine the inhibition of cell growth for several different celllines (HT-29: human colon rumor, MCF-7: human breast tumor, B16: murinemelanoma, P388: murine leukemia, P388/CPT: CPT-resistant murine leukemiacells) using the MTT assay.

The in vitro effectiveness of TPT correlates well with its usefulness invivo. In particular, the topoisomerase I IC₅₀ for TPT-HCl was 0.504μg/mL (1.1 μM, MW=457.9 g/mol). See Wall et al., "Plant AntitumorAgents. 30. Synthesis And Structure Activity Of Novel CamptothecinAnalogs," J. Med. Chem. 36:2689 (1993)!. In side by side in vivo studiesTPT was superior to unmodified camptothecin against P388 murineleukemia, Lewis lung carcinoma, and B16 murine melanoma. See Johnson etal., "Preclinical Profile Of SK And F 104684, A Water-Soluble Analog OfCamptothecin," Presented At The Sixth NCI-EORTC Symposium On New DragsIn Cancer Therapy, Amsterdam, March, 1991 and Johnson et al.,"Comparative Efficacy Of Topotecan, Irenotecan, Camptothecin And9-aminocamptothecin In Preclinical Tumor Models," In Proceedings On TheSeventh NCI-EORTC Symposium On New Drags In Cancer Therapy, Amsterdam,1992, p. 85!.

In contrast, the in vitro efficacy for CPT-11 is negligible seeKingsbury et al. supra and Kawato et al. supra! and belies its in vivoeffectiveness. Researchers have believed that the lack of topoisomeraseI inhibitory activity of CPT-11 is due to the fact that CPT-11 must bemetabolized by the body into a bioreactive form, (SN-38), thus making ita `pro-drug`.

II. Properties Of Camptothecin Analogs Of The Present Invention

A. Physical Properties

The camptothecin analogs of the present invention have significantlyimproved water solubility compared to modified camptothecin as shown inTable 1. The improved solubility is due to the novel carbohydrate groupsattached to the relatively hydrophobic camptothecin ring system. Notethat the modified camptothecin analogs of the present invention will bereferred hereinafter by their abbreviated codes as follows: HAR4=7-4,6-di-O-acetyl-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin;HAR5=7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin;HAR6=7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin;HAR7=7-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin.

                  TABLE 1                                                         ______________________________________                                        Analog      Solubility (μg/mL)                                             ______________________________________                                        HAR4        155                                                               HAR5        35                                                                HAR6        1620                                                              HAR7        62                                                                ______________________________________                                    

In addition to testing for water solubility, one of the analogs of thepresent invention, HAR7, was tested for solubility in various othersolvents and solvent mixtures, to determine the system which providesthe greatest solubility for the analog. The results of those solubilityexperiments are shown below in Table 2:

                  TABLE 2                                                         ______________________________________                                        Solvent           Solubility (μg/ml)                                       ______________________________________                                        5%       Ethanol/DSW.sup.1                                                                          177                                                     5%       Ethanol/Water                                                                              80                                                      10%      Ethanol/Water                                                                              143                                                     20%      Ethanol/Water                                                                              380                                                              Pure Ethanol 1983                                                    0.1      M Citrate Buffer                                                                           24                                                      0.2      M Acetate Buffer                                                                           23                                                      2        M Acetate Buffer                                                                           29                                                      0.2      M NaH.sub.2 PO.sub.4 Buffer                                                                32                                                      0.2      M Na.sub.2 HPO.sub.4 Buffer                                                                30                                                      4:1:5    PG.sup.2 /Ethanol/Water                                                                    740                                                     4:1:5    PG/Ethanol/Saline                                                                          893                                                     4:1:5    PG/Ethanol/DSW                                                                             960                                                              PEG 300.sup.3                                                                              >5000                                                   ______________________________________                                         Key:                                                                          1) DSW.                                                                       2) PG: Propylene Glycol.                                                      3) PEG 300: Polyethylene Glycol 300.                                     

III. Synthesis Of Novel Glycosylated Camptothecin Analogs

A recent development in the area of pharmaceutical science has centeredaround efforts to increase to bioavailability of known drugs by chemicalderivatization. See L. Brown and R. Thomas, Aust. J. Pharm. Sci. 8:1(1979); Y. H. Ji et al., J. Med. Chem. 33:2264 (1990); V. Stella et al.,J. Med. Chem. 35:145 (1992); and Kleeman et al., J. Med. Chem. 35:559(1992)!. One approach used by researchers is to develop methods ofglycosylating a variety of medicinally-important compounds with theobjective of increasing aqueous solubility while hopefully enhancing thepharmacological profile of these agents. Such a process could unlock thebenefits of a broad array of biologically-active compounds withintrinsically modest hydrophilicity.

The chemistry of glycals is perfectly suited for addressing the aboveissues. Glycals, cyclic sugar derivatives containing a 1,2-double bond,are indispensable synthetic precursors in the field of carbohydratechemistry. Though this class of sugars was discovered by Fischer 80years ago see E. Fischer and K. Sitzungsber, Preuss. Akad. Wiss. 16:311(1913)!, there has recently been an immense volume of research usingthese compounds to synthesize complex polysaccharides and glycosylatedproducts.

One reaction in particular, discovered by Ferrier in 1969 see R. J.Ferrier, J. Chem. Soc. C p. 570 (1969)! which described a reaction whichglycals could be attached to various nuclcophiles, allowed syntheticchemists to attach carbohydrates to a variety of non-carbohydrateorganic molecules. The result compound being an O-glycoside in which acarbohydrate moiety is attached to an oxygen atom of a typicallyhydrophobic aglycon (aglycon referring to the non carbohydrate molecule)unit. Although similar glycosylation reactions had been accomplishedthermally using water, alcohols and phenols see B. Helferich, Adv.Carbohydrate Chem. 7:209 (1952); R. J. Ferrier, J. Chem Soc. p. 5443(1964); and R. J. Ferrier et al., J. Chem Soc. p. 3667 (1962)!, theFerrier reaction's use of boron trifluoride etherate greatly expandedthe synthetic scope of the reaction.

Despite its synthetic utility, the Ferrier reaction has been lesssuccessfully applied to the commercial glycosylation of medicinallyuseful compounds. Such reactions, preferably performed on a large scale,require the use of Lewis acid catalysts which are more efficient, lesstoxic, and less destructive toward the aglycon to be glycosylated. Forexample, since most of these strong Lewis acids spontaneously react withair and moisture, the use of these Lewis acids presents serious problemsin their handling, particularly under the large-scale, industrialsetting. Another approach to glycosylation that employs a glycalderivative requires the use of expensive metal catalyst whose effects tohuman health presents serious drawbacks. See Hacksell, U., Daves, G. D.,Jr., J. Org. Chem. 48:2870 (1983)!. For these reasons, the use of thenon-toxic, stable catalyst iodine see U.S. Pat. No. 5,278,296, herebyincorporated by reference!, which is an extremely mild Lewis acid andyet according to the invention retains enough acidity to effectglycosylation, is the preferred reagent.

In one preferred aspect, the invention concerns O-glycoside compoundsobtained by reacting either a soft carbon or oxygen nucleophile compoundand a glycosylating agent selected from 3-acylated five- andsix-membered glycals in the presence of a catalytic amount of iodine(5-50 mol % with 20 mol % being the most representative) to provide areaction mixture containing the glycosylated product.

The present invention contemplates the preparation of the necessaryglycals by the following procedure. First, the desired carbohydrate maybe obtained commercially in non-acetylated form and acetylated byreaction with acetic anhydride in acetic acid with a catalytic amount ofhydrobromic acid. The acetylated carbohydrate is thereafter converted toan acetylated carbohydrate halide (e.g., bromide, by reaction withhydrobromic acid in acetic acid). The acetylated carbohydrate halide isconverted to the acetylated glycal by reaction with Zn/CuSO₄. Finally,the acetylated glycal is converted into a more reactive diacetyl benzoylglycal form by reaction with ortho-anisic acid (2-methoxybenzoic acid).These glycals, which are considered derivatizing agents, are thenreacted with a modified form of camptothecin, namely 7-hydroxymethylcamptothecin. This modified form of camptothecin is itself synthesizedby the reaction of camptothecin with a modifying reagent of methanol.Alternatively, a commercially available acetylated carbohydrate may beused and thereby obviate the need for the initial reaction step.

For glycosylation, preferred glycals of the formulas I-III and Ia-IIIaare illustrated in FIG. 17, where R₀ is a lower alkyl group and R₁, R₂and R₃ are the same or different and represent an aliphatic acyl groupor an aromatic acyl group such as a benzoyl group.

Any of various suitable solvents can be used for the glycosylationreaction of which THF, acetone, diethyl ether, methylene chloride,chloroform, and benzene are preferred. The reaction temperature and timecan be varied, e.g., ranging from -78° to room temperature for about 0.5to 12 hours.

In another preferred aspect, the invention concerns partly andcompletely deacylated products having enhanced water-solubility,produced by hydrolysis of one or more acyl groups from the acylatedproduct, under per se commonly used conditions for hydrolysis andworkup, namely with Zn (OAc)₂ o 2H₂ O in methanol or ammonia inmethanol.

IV. Methodology For Screening Camptothecin Analogs

We claim chemotherapeutic anti-cancer glycosylated analogs of thecamptothecin molecule. These compounds will most often be made by theglycosylation of a camptothecin analog. This section outlines procedureswhereby a novel glycosylated camptothecin analog can be screened foruseful biological activity.

SCREENING PROCEDURE A

Screening Mode A consists of the following biological tests:

Mode I: Determine whether the aglycone unit of the glycosylatedcamptothecin analog of interest possesses at least one primary orsecondary hydroxyl functional group which can be glycosylated with thesugar unit of interest.

Mode II: Determine whether the glycosylated camptothecin analog ofinterest has equal or better aqueous solubility than unmodifiedcamptothecin (i.e., ≧20 μg/mL).

Mode III: Determine whether the glycosylated camptothecin analog ofinterest or the corresponding aglycon inhibits Topoisomerase I, therebyhalting DNA replication, no less than 10 times worse than unmodifiedcamptothecin (i.e., has a molar IC₅₀ no less than 10 times lower thanthat of unmodified camptothecin) and can be called a topoisomeraseinhibitor.

Mode IV: Determine whether the glycosylated camptothecin analog ofinterest inhibits in vivo carcinoma growth by the use of animal models,and is therefore an in vivo carcinoma growth inhibitor.

Mode V: Determine whether the glycosylated camptothecin analog ofinterest exhibits animal toxicity equal to or less than unmodifiedcamptothecin.

A new camptothecin analog ("X") can be evaluated for biological activityusing the procedure outlined in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Evaluation Of Biological Activities Of Novel Camptothecin Analogs             Mode                                                                             Result                                                                            Interpretation/Next Step                                               __________________________________________________________________________    I  +react                                                                            Glycosylation of aglycone unit of analog is readily carried out.              Perform                                                                       glycosylation reaction and evaluate in Modes II, III, IV, and V.          -react                                                                            Glycosylation of aglycone unit of analog is not readily carried               out and                                                                       should not be evaluated further.                                       II +solub                                                                            Glycosylated compound is water soluble/Evaluate in Modes III, IV,             and V.                                                                    -solub                                                                            Glycosylated compound is not water soluble and is not useful as an            anti-                                                                         cancer chemotherapeutic agent. No further evaluation necessary.        III                                                                              +inhib                                                                            Glycosylated compound or its aglycon is a Topoisomerase I                     inhibitor/Evalulate in Modes IV and V.                                    -inhib                                                                            Glycosylated compound is not a Topoisomerase I inhibitor.              IV +inhib                                                                            Glycosylated compound is an in vivo carcinoma growth                          inhibitor/Evaluate                                                            in Mode V to determine animal toxicity.                                   -inhib                                                                            Glycosylated compound is no an in vivo carcinoma growth inhibitor             and is                                                                        not useful as an anti-cancer chemotherapeutic agent.                   V  +toxic                                                                            Glycosylated compound exhibits animal toxicity greater than                   camptothecin                                                                  and is not useful as an anti-cancer chemotherapeutic agent.               -toxic                                                                            Glycosylated compound does not exhibit animal toxicity greater                than                                                                          camptothecin and is useful as an anti-cancer chemotherapeutic                 agent.                                                                 __________________________________________________________________________     Key:                                                                          +react = glycosylation reaction possible; -react = glycosylation reaction     impossible; solub = water soluble; -solub = not water soluble; +inhib =       process inhibited; -inhib = process not inhibited; +toxic = exhibits          greater animal toxicity than camptothecin; and -toxic = exhibits equal or     less animal toxicity than camptothecin.                                  

V. In Vivo Uses

The present invention contemplates using therapeutic compositions ofsoluble camptothecin analogs, and in particular for treatment of cancer.It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients. Inaddition, camptothecin analogs may be used together with otherchemotherapeutic agents, including unmodified camptothecin.

With respect to the mode of administration, the camptothecin analogs maybe employed for intravenous, intramuscular, intrathecal or topical(including topical ophthalmic) administration. Formulations for suchadministrations may comprise an effective amount of camptothecin analogin sterile water or physiological saline.

On the other hand, formulations may contain such normally employedadditives as binders, fillers, carriers, preservatives, stabilizingagents, emulsifiers, buffers and excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions typically contain 1%-95% of active ingredient, preferably2%-70%.

The compositions are preferably prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared.

The camptothecin analogs of the present invention are often mixed withdiluents or excipients which are compatible and physiologicallytolerable. Suitable diluents and excipients are, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired the compositions may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, stabilizingor pH buffering agents.

Options for optimal method of camptothecin analog administrationinclude, but are not limited to: a 30-minute infusion every three weeks,a 30-minute infusion daily×5 every three weeks, a 24-hour infusion everythree weeks, a 120-hour infusion every three weeks, and a 72-hourinfusion repeated every three weeks.

Likewise, dosage ranges for camptothecin analog treatment include, butare not limited to: 1 to 200 mg/kg/day.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: PBS (phosphate buffered saline); MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide); EDTA(ethylenedinitrotetraacetic acid disodium salt); HCl (hydrogenchloride); Tris (triphenylphosphine); NaCl (sodium chloride); SDS(sodium dodecyl sulfate); Na₂ SO₂ O₃ (sodium thiosulfate); TAE(Tris-Acetate-EDTA); H₂ SO₄ (sulfuric acid); FeSO₄ (ferrous sulfate);CuSO₄ (cuprous sulfate); MgSO₄ (magnesium sulfate); NaOAc (sodiumacetate); DMF (dimethyl formamide); THF (tetrahydrofuran); NaHCO₃(sodium bicarbonate); HBr (hydrogen bromide); KBr (potassium bromide);DMSO (dimethyl sulfoxide); DMSO-d₆ (fully deuterated dimethylsulfoxide); CHCl₃ (chloroform); CDCl₃ (deuterated chloroform); NH₃(ammonia); IMEM (Iscove's Minimum Essential Medium); IMDM (Iscove'sModified Dulbecco's Medium); D-MEM (Dulbecco's Modified Eagle Medium);HEPES ((N- 2-hydroxyethyl!piperazine-N'- 2-ethanesulfonic acid!));AntiPPLO (Antibody against Pleuropneumonia-like Organism); pm (parts permillion); α! (specific rotation); μL (microliters); μg (micrograms); mL(milliliters); L (liters); mg (milligrams); g (grams); hr (hours); mM(millimolar); μM (micromolar); nM (nanomolar); N (normal); nm(nanometers); min (minutes); IU (intravenous units); s.c.(subcutaneous); mm (millimeter); MTD (maximally tolerated dosage); i.p.(intraperitoneal); kg (kilograms); δ(chemical shift); J (couplingconstant); s (singlet); d (doublet); t (triplet); q (quartet) m(multiplet); vs (very strong); s (strong); m (medium); w (weak); vw(weak); v (variable); mp (melting point); c (optical path length); NMR(Nuclear Magnetic Resonance); IR (Infrared Spectroscopy); MHz(megahertz); Hz (hertz); cm⁻¹ (wavenumbers).

EXAMPLE 1 Solubility Determination Of Both Unmodified Camptothecin AndCamptothecin Analogs

The water solubility of the camptothecin analogs of the presentinvention were measured in phosphate buffered saline, pH 7.5 (PBS) by aspectrophotometric assay. Approximately 5 mg of test compound wassuspended in 1 mL of PBS in a 1.5 mL cryovial. The suspensions weremixed continuously on a Thermolyne vari-mix at room temperature for 24hr. The suspensions were then centrifuged at 14000× g for 2 minutes toseparate the undissolved materials. The supernatant fluids were dilutedappropriately with PBS and the ultraviolet spectrums were recorded on aBeckman 640 DU spectrophotometer. For comparison, a standard stock (2mg/mL in DMSO) solution was prepared for each analog. The ultravioletspectrum of a 20 of 40 μg/mL standard working solution was measured. Thewater solubility of each analog was calculated based on the followingformula:

    C.sub.unknown =C.sub.standard ×(A.sub.unknown ×D.sub.dilution)/A.sub.standard

where C_(unknown) is the concentration of the unknown solution to bedetermined; C_(standard) is the concentration of the standard workingsolution (either 20 or 40 μg/mL); A_(standard) is the absorbance at 350nm of the standard working solution; D_(Dilution) is the appropriatedilution factor used so that the absorbance of the unknown workingsolution is within the dynamic range of the UV spectrophotometer (lessthan 2 absorbance units).

The solubility of HAR7 in various solvents and solvent systems wasmeasured using one of two experimental methods, depending on theconditions and solvents used.

First, an evaporation method was used in which an excess quantity ofHAR7 was added to the solvent of choice. The mixture was finely dividedusing ultrasonic vibration in order to dissolve the material thoroughly.The resulting mixture was filtered and the solution was evaporated todryness using a rotary evaporator.

Second, a spectrophotometric method was used in which an excess quantityof HAR7 was added to the solvent of choice. The mixture was finelydivided using ultrasonic vibration in order to dissolve the maximumamount of solid. The resulting suspension was filtered through a 0.45 μmnylon filter, and the clear solution was diluted in the same solvent andits concentration was measure spectrophotometrically, based upon anextinction coefficient of 17000 at 258 nm.

EXAMPLE 2 Determination Of Biological Activity For Both UumodifiedCamptothecin And Camptothecin Analogs

A) Cell Culture Preparation

Murine B16 melanoma cell line was grown in RPMI 1640 medium supplementedwith 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50units/mL penicillin, 50 μg/mL streptomycin, 25 μg/mL gentamicin, 0.75%sodium bicarbonate 10 mM HEPES buffer (pH 7.4), and 0.06 mg/mL AntiPPLO.Murine P388 leukemic cell line and human HT-29 colon adenocarcinoma linewere maintained in RPMI 1640 medium supplemented with 10%heat-inactivated fetal bovine serum. P388/CPT (camptothecin resistantcell line) was maintained in RPMI 1640 medium supplemented with 20%heat-inactivated fetal bovine serum, 10 μM β-mercaptoethanol, 10 mML-glutamine, 100 IU/mL streptomycin, and 50 μg/mL gentamicin. MCF-7Mhuman breast adenocarcinoma was maintained in IMEM supplemented with 5%non heat-inactivated fetal bovine serum and 1 nM insulin.

B) Determination Of In Vitro Growth Inhibitory Activity

The biological assay used to test the in vitro effectiveness of thecamptothecin analogs of the present invention was first described byMosmann in 1983. See "Rapid Colorimetric Assay For Cellular Growth AndSurvival: Application To Proliferation And Cytotoxicity Assays," J.Immun. Meth. 65:55 (1983)!. The assay utilizes a tetrazolium salt toquantitatively measure mammalian cell survival and proliferation bycolorimetric methods. In particular, MTT, a pale yellow compound withminimal absorbance, is incubated with cancerous cells in addition to (orin the absence of) a particular camptothecin analog. Living cells withactive mitochondrial enzymes metabolize the MTT into a dark blue/purpleformazan product with high absorbance. The exact procedure used to testthe camptothecin analogs of the present invention is described below.

Exponentially growing cells (1-2×10³ cells, unless specified otherwise)in 0.1 mL medium were seeded on day 0 in a 96-well microtiter plate. Onday 1, 0.1 mL aliquots of medium containing graded concentrations oftest analogs were added to the cell plates. After incubation at 37° C.in a humidified incubator for 3 days (P388, P388/CPT, B16) or 6 days(HT-29, MCF-7M), the plates were centrifuged briefly and 100 μL of thegrowth medium was removed. Cell cultures were incubated with 50 μL of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide MTT, 1mg/mL in Dulbecco's phosphate buffered saline (PBS)! for 4 hr at 37° C.The resulting purple formazan precipitate was solubilized with 200 μL of0.04N HCl in isopropyl alcohol. Absorbance was monitored in a BioRadModel 3550 Microplate Reader at a test wavelength of 570 nm and areference wavelength of 630 nm. The absorbance is transferred to a PC486 computer. The IC₅₀ values were determined by a computer program(EZ-ED50) that fits all of the data to the following four-parameterlogistic equation:

    Y=(A.sub.max -A.sub.min)/(1+ X/IC.sup.50 !).sup.n +A.sub.min

where A_(max) is the absorbance of control cells, A_(min) is theabsorbance of cells in the presence of highest agent concentration, IC⁵⁰is the concentration of agent that inhibits the cell growth by 50% ofcontrol cells (based on the absorbance) and n is the slope of the curve.

For the in vitro cell inhibition assays, camptothecin was found to havethe following IC₅₀ (50% cell growth inhibition concentration) as shownin Table 4:

                  TABLE 4                                                         ______________________________________                                               Cell Line                                                                            IC.sub.50 (μg/mL)                                            ______________________________________                                               HT-29  0.002                                                                  MCF-7  0.001                                                                  B16    0.015                                                                  P388   0.010                                                                  P388/cpt                                                                             1.123                                                           ______________________________________                                    

The camptothecin analogs of the present invention display strong invitro anti-cancer effectiveness. As shown in Table 5, the analogs of thepresent invention show in vitro activity against several of thecancerous cell lines described above.

                  TABLE 5                                                         ______________________________________                                        Growth Inhibitory Activity IC.sub.50 (μg/mL)                               Analog  P388     P388/cpt B16    HT29 MCF-7                                   ______________________________________                                        HAR4    0.03     7.04     0.026  0.011                                                                              0.0027                                  HAR5    0.362    21.2     0.43   0.105                                                                              0.01                                    HAR6    1.68     28.4     2.43   0.476                                                                              0.111                                   HAR7    0.088    18.7     0.173  0.031                                                                              0.0064                                  ______________________________________                                    

C) Inhibition Of Topoisomerase I Catalytic Activity

The topoisomerase I catalytic activity was measured by convening thesupercoiled SV40 DNA (Form I) to the relaxed form (Form I_(o)). Allreactions were performed in 20 μL reaction buffer (Tris-HCl, 10 mM, pH7.5; EDTA, 1 mM; NaCl, 100 mM) with 0.25 μg SV40 DNA, 0.5 unit of humanplacental topoisomerase I (TopoGen) and graded concentrations of theanalog tested. The reaction mixtures were incubated at 37° C. for 30minutes. The topoisomerase I activity was stopped by incubating thereaction mixture with 1 μL of 10% SDS and 1 μL of proteinase K (1.25mg/mL) for additional 30 min. One μL of the loading buffer (1%bromophenol blue and 48% sucrose) was then added. Ten μL of the reactionmixture was loaded onto a 1% agarose gel prepared in TAE buffercontaining 2 μg/mL chloroquine; and the electrophoresis was performed at82 volt for 4.5 hr in the TAE buffer containing 2 μg/mL chloroquine.Chloroquine is added to separate nicked and relaxed DNA molecules;without chloroquine, the fully relaxed Form I_(o) comigrated with thenicked DNA. The gels were then stained with 0.5 μg/mL ethidium bromidesolution for 30 min or longer (if chloroquine is present during theelectrophoresis step), and destained with 5 changes of deionized water.DNA bands were visualized with a 254 nm ultraviolet light (SpectrolineTransilluminator Model TL-254A) and documented with a Polaroid 665positive/negative instant pack film. The DNA bands (image) on thenegative were densitometrically scanned with a Molecular DynamicPersonal Densitometer. The percent inhibition of Toposiomerase Iactivity is calculated based on the following equation:

    % Inhibition=(F.sub.SC(E+D) -F.sub.SC(E))/(F.sub.SC(C) -F.sub.SC(E))×100

where F_(SC)(E+D) represents fraction of supercoiled DNA in the presenceof enzyme and drug; F_(SC)(E) represents fraction of supercoiled DNA inthe presence of enzyme alone; F_(SC)(C) represents fraction ofsupercoiled DNA in the untreated SV40 DNA; the IC₅₀ value was estimatedusing the same four-parameter logistic equation described in the invitro growth inhibition studies.

The growth inhibitory activity of camptothecin (HAR1) and 6 analogs ofthe present invention against the P388, P388/CPT (camptothecin resistantcell line), B16, H-29, and MCF-7M tumor cells was determined using a MTTassay.

The inhibition of topoisomerase I catalyzed relaxation of supercoiledSV40 DNA was determined by separation of supercoiled DNA by camptothecinand the six analogs of the present invention is shown in FIGS. 18 and19. The growth inhibitory activity expressed as IC₅₀ values(concentration of agents inhibits the growth of the cells by 50% of thecontrol cells)!, and the inhibitory activity of topoisomerase I alsoexpressed as IC₅₀ values (concentrations of agents which inhibits thetopoisomerase I activity by 50% of the control)! of these camptothecinanalogs are summarized in Tables 5 and 6.

Two different modifications were made on the camptothecin molecule. Inthe first modification, the 20S hydroxy group of the lactone ring (ringE) of camptothecin was converted into acetate (HAR3) or hexanoate(HAR2). The water solubility decreased from 20 μg/mL for camptothecin(HAR1) to 6.5 and 2.9 μg/mL, for the acetate (HAR3) and the hexanoate(HAR2), respectively. The growth inhibitory activity was reducedapproximately 10 and 100 fold, respectively for the acetate (HAR3) andthe hexanoate (HAR2). Neither the acetate (HAR3) nor the hexanoate(HAR2) inhibited the topoisomerase I activity even at the highestconcentration tested (100 μg/mL). Therefore, the 20S hydroxyl group isessential for the biological activity (i.e., inhibition of TopoisomeraseI activity, and growth inhibition activity).

In the second modification, either a monosaccharide camptothecin glucalacetate, HAR4, and camptothecin hydroxyl glucal, HAR7! or disaccharidecamptothecin maltal acetate, HAR5, and camptothecin hydroxyl maltal,HAR6! was attached to the 7-hydroxymethyl group of camptothecin. Thewater solubility of camptothecin hydroxyl glucal and camptothecinhydroxyl maltal improved 16- and 80-fold, respectively.

The growth inhibitory activity and topoisomerase I inhibitory activityof camptothecin glucal acetate (HAR4) and camptothecin hydroxy glucal(HAR7) are quite similar to those of camptothecin. These data suggestthat biological activity is retained or even enhanced if the sevenposition of camptothecin is appropriately substituted. It is of interestto note that camptothecin glucal acetate (HAR4) is 2-3 fold more potentin inhibiting the growth of tumor cells than camptothecin hydroxylglucal (HAR7). However, camptothecin hydroxyl glucal (HAR7) is 10-timesmore inhibitory than camptothecin glucal acetate (HAR4) in thetopoisomerase I assay. The reason for this disparity is unknown. It ispossible that camptothecin glucal acetate (HAR4) is taken up morerapidly into cells because of greater lipophilicity. Other assays tomeasure trapping of enzyme cleavable complexes by these analogs might beneeded to fully characterize their effects on topoisomerase I.

The growth inhibitory activity is further reduced if maltal is asubstituent on the 7-hydroxymethyl group of camptothecin. A similartrend was noted as in the glucal series; camptothecin maltal acetate(HAR5) was more cytotoxic than camptothecin hydroxyl maltal (HAR6). Bothcompounds moderately inhibited topoisomerase I activity at the highestconcentration (100 μg/mL) tested. Thus, camptothecin hydroxyl maltal(HAR6) may be a good candidate as a prodrug of7-hydroxymethylcamptothecin. However, it remains to be determinedwhether this compound is converted into an "active species" in animalsor humans.

All of the six analogs are cross-resistant to camptothecin as indicatedin the differential growth inhibitory activity against the parent andcamptothecin resistant cell lines. These data provide further supportthat these agents have similar mechanism as that of camptothecin.

Camptothecin was found to have a topoisomerase I IC₅₀ (50% activityinhibitory concentration) of 8.0 μg/mL.

The camptothecin analogs of the present invention display strong invitro anti-cancer effectiveness. As shown in Table 6 the analogs of thepresent invention show significant Topoisomerase I inhibitory activity.

                  TABLE 6                                                         ______________________________________                                        Analog     Topoisomerase I IC.sub.50 (μg/mL)                               ______________________________________                                        HAR4          25                                                              HAR5       >100                                                               HAR6       >100                                                               HAR7          2                                                               ______________________________________                                    

D) In Vivo Animal Testing of Camptothecin Analogs

In vivo animal testing was performed on the camptothecin analogs of thepresent invention using mice and five experimental tumor models; namelythe murine P388 leukemia, the murine B16 melanoma, the MX-1 human breasttumor xenograft, the human lung tumor xenograft, and the human prostatetumor xenograft. For all the models, the vehicle used to deliver theanalog of interest was isotonic saline.

1) Murine B16 Melanoma Model

For the B16 melanoma model, the following procedure was used. B6D2F1mice receive i.p. inocula of B16 murine melanoma brei prepared from B16tumors growing s.c. in mice (day 0). On day 1, tumored mice are treatedwith drugs of vehicle control; the route of drug administration andschedule are selected as appropriate for the study in question. Ifdosing information for agents is not available, the maximum tolerateddose (MTD) is determined in initial dose finding experiments innon-rumored mice. In a typical experiment, drugs are given at their MTDand 1/2 MTD doses i.p. on a daily×5 schedule.

The mean survival times of all groups are calculated, and results areexpressed as mean survival of treated mice/mean survival of control mice(T/C)×100%. A T/C value of 150 means that the mice in the treated grouplived 50% longer than those of the control group; this is sometimesreferred to as the increase in life span or ILS value.

Mice that survive for 60 days are considered long-term survivors, orcures, in the B16 model. The universally accepted cut-off for activityin this model, which has been for years by the National CancerInstitute, is T/C=125. Conventional use of B16 over the years has setthe following levels of activity: T/C<125, no activity, T/C=125-150,weak activity; T/C=150-200, modest activity; T/C=200-300, high activity;T/C>300, with long term survivors; excellent, curative activity.

The results for the B16 melanoma model are shown below in Table 7:

                  TABLE 7                                                         ______________________________________                                                                    Tox                                               Analog  Dose       Survivors                                                                              Deaths                                                                              T/C.sup.1                                                                          ΔWeight (%)                      ______________________________________                                        Negative                                                                              N/A.sup.1  0/10     0/10  100% +11.3%                                 Control                                                                       (Saline)                                                                      Positive                                                                              4      mg/kg   0/10   0/10  212% -7.0%                                Control 2      mg/kg   1/10   0/10  201% +1.9%                                (TPT)                                                                         HAR 6   150    mg/kg   0/10   7/10  185% -27.2%                                       100    mg/kg   0/10   1/10  184% -13.8%                               HAR 4   20     mg/kg   1/10   1/10  212% -15.6%                                       10     mg/kg   1/10   0/10  189% +6.6%                                HAR 5   200    mg/kg   3/10   0/10  232% +5.9%                                        100    mg/kg   3/10   0/10  239% +9.0%                                HAR 7   40     mg/kg   1/10   0/10  205% -7.9%                                        20     mg/kg   0/10   0/10  191% -0.1%                                ______________________________________                                         Key:                                                                          1) N/A = Not Applicable.                                                 

The high dose (150 mg/kg) of HAR 6 was lethal to mice, causing thedeaths of 7/10 animals in the group. The lower dose of 100 mg/kg, provedto be the MTD, causing approximately 14% weight loss in the mice, withone drug-related death. HAR 6 administered at 100 mg/kg i.p. on a fiveday schedule gave a T/C value of 184 with no cures. Topotecan at dosesof a 4 mg/kg and 2 mg/kg achieved T/C values of 213 and 210respectively; one 60-day survivor was observed with the 2 mg/kg dose.

Of the four analogs (including HAR 6), HAR 5 demonstrated the highestefficacy vs. B16; three 60-day survivors ("cures") each were obtained atdoses of 200 mg/kg and 100 mg/kg respectively (daily×5 schedule). T/Cvalues for the remaining mice of 232 and 239 were achieved at the highvs. the low dose of HAR 5. HAR 4 produced one cure and a T/C=212 at itsMTD of 20 mg/kg, and also gave one cure and a T/C=189 at the lower doseof 10 mg/kg (qd×5). Treatment with HAR 7 also resulted in one 60-daysurvivor and a T/C value of 191 (no cures). All three compounds werewell-tolerated at their MTDs, with acceptable weight loss and only onetoxic death (HAR 4 high dose group).

2) Murine P388 Leukemia Model

For the P388 leukemia model, the procedure followed is exactly the sameas the aforementioned B16 model. The tumor inoculam is prepared byremoving ascited fluid containing P388 cells from tumored B6D2F1 mice,centrifuging the cells, and then resuspending the leukemia cells insaline. Mice receive 1×10⁶ P388 cells i.p. on day 0.

The results for the P388 leukemia model are shown below in Table 8:

                  TABLE 8                                                         ______________________________________                                                                    Tox                                               Analog  Dose       Survivors                                                                              Deaths                                                                              T/C.sup.1                                                                          ΔWeight (%)                      ______________________________________                                        Negative                                                                              N/A.sup.1  0/10     0/10  100% +17.9%                                 Control                                                                       (Saline)                                                                      Positive                                                                              4      mg/kg   3/10   0/10  297% -6.6%                                Control                                                                       (TPT)                                                                         HAR 4   20     mg/kg   2/10   0/10  206% -14.0%                                       10     mg/kg   2/10   0/10  201% -0.6%                                HAR 5   200    mg/kg   0/10   0/10  161% -2.0%                                        100    mg/kg   1/10   0/10  141% +0.7%                                HAR 6   100    mg/kg   3/10   0/10  320% -12.2%                                       50     mg/kg   1/10   0/10  225% -3.5%                                HAR 7   40     mg/kg   3/10   0/10  223% -16.0%                                       20     mg/kg   0/10   0/10  192% -1.7%                                ______________________________________                                         Key:                                                                          1) N/A = Not Applicable.                                                 

The four Harrier topoisomerase I inhibitors were evaluated via i.p.administration in the same experiment vs. the murine P388 leukemia;topotecan was again included as the positive drug control. HAR 4, 6 and7 all demonstrated high, curative efficacy on a five day schedule vs.the P388 leukemia, even though the agents diffused considerably in theirpotencies. HAR 6 at its MTD of 100 mg/kg produced the best antileukemicresult, causing three 30-day survivors ("cures") and an impressive T/Cvalue of 320 for the remaining seven mice. The lower dose of 100 mg/kgwas less effective, but still highly active (T/C=225, with one cure).

HAR 4 and 7 also demonstrated excellent activity vs. P388. HAR 4 at 20and 10 mg/kg produced T/C values of 206 and 201 respectively; two cureswere also achieved at each dose. HAR 7 at doses of 40 and 20 mg/kg gaveT/C values of 223 and 192 respectively, with three 30-day survivorsrecorded at the high dose. HAR 5 was less efficacious compared to theother three compounds, although one cure was obtained at the low dose of100 mg/kg.

HAR 4, 6 and 7 were tested at their MTDs vs. P388 leukemia, as evidencedby a 12%-16% weight loss for these agents at the high dose (no toxicdeaths occurred). HAR 5 was tested at its solubility-limiting dosebecause only insignificant weight loss was recorded for this analog.

Topotecan at its MTD of 4 mg/kg (daily×5) demonstrated a high degree ofefficacy, comparable to that produced by HAR 6. Three mice treated withtopotecan were recorded as 30-day survivors, and the remaining 7 animalsexperienced a T/C=297.

3) MX-1 Human Breast Tumor Model

For the MX-1 human breast tumor xenograft models, the followingprocedure was used. Nude mice are implanted s.c. by trocar withfragments of MX-1 mammary carcinomas harvested from s.c. growing MX-1tumors in nude mice hosts. When tumors are approximately 5 mm×5 mm insize (usually about ten days after inoculation), the animals arepair-matched into treatment and control groups. Each group contains tentumored mice, each of which is ear-tagged and followed individuallythroughout the experiment. The administration of drugs or vehicle beginsthe day the animals are pair-matched (day 1). The doses, route of drugadministration and schedule are selected as appropriate for the study inquestion. If the MTD dose of an agent is not known, it is determined inan initial dosing experiment in non-tumored mice. In a typicalexperiment, drugs are given at their MTD and 1/2 MTD doses i.p. on adaily×5 schedule.

The experiment is usually terminated when control tumors reach a size of2-3 g. Mice are weighed twice weekly, and tumor measurements are takenby calipers twice weekly, starting on day 1. These tumor measurementsare converted to mg tumor weight by a well known formula, and from thesecalculated tumor weights the termination date can be determined. Upontermination, all mice are weighed, sacrificed, and their tumors excised.Tumors are weighed, and the mean tumor weight per group is calculated.In this model, the mean treated tumor weight/mean control tumorweight×100% (T/C) is subtracted from 100% to give the tumor growthinhibition for each group.

Some drugs cause tumor shrinkage in the MX-1 model. With these agents,the final weight of a given tumor is subtracted from its own weight atthe start of the treatment on day 1. This difference divided by theinitial tumor weight is the % shrinkage. A mean % tumor shrinkage can becalculated from data from the mice in a group that experienced MX-1regressions. If the tumor completely disappears in a mouse, this isconsidered a complete regression or complete tumor shrinkage. Ifdesired, mice with partial or total tumor regressions can be kept alivepast the termination date to see whether they live to become long term,tumor-free survivors.

The camptothecin analogs of the present invention display strong in vivoanti-cancer effectiveness as well. As shown in Table 9, the analogs ofthe present invention show in vivo activity against MX-1 human breastxenografts implanted in mice. Note that in the following table theabbreviation SR refers to tumor shrinkage rate, while those entriesfollowed by a superscripted `2` are tumor growth inhibition rates, nottumor shrinkage rates.

                  TABLE 9                                                         ______________________________________                                                           Tox     Shrink-                                            Analog  Dose       Deaths  age   SR (%).sup.1                                                                        ΔWeight (%)                      ______________________________________                                        Negative                                                                              N/A        0/10    0/10   0%    +8.7%                                 Control                                                                       (Saline)                                                                      Positive                                                                              4      mg/kg   0/10  6/10  94%   -15.4%                               Control                      1/10  100%                                       (Topotecan                                                                    HAR 6   50     mg/kg   0/10  8/10  81%    +2.5%                                       100    mg/kg   1/10  9/10  94%   -18.6%                               HAR 4   10     mg/kg   0/10  8/10  77%   -10.2%                                                            2/10  100%                                               20     mg/kg   9/10  1/10  99%   -31/2%                               HAR 5   100    mg/kg   0/10  10/10 .sup. 48%.sup.2                                                                      +7.4%                                       200    mg/kg   0/10  3/10  73%    +5.3%                                                            7/10  .sup. 82%.sup.2                            HAR 7   20     mg/kg   0/10  10/10 98%   -0.05%                                       40     mg/kg   4/10  5/10  98%   -25.0%                               ______________________________________                                         Key:                                                                          1) SR = Tumor Shrinkage Rate.                                                 2) Tumor growth inhibition rate, not tumor shrinkage rate.               

The results demonstrate that HAR 6 has impressive antitumor activity vs.MX-1. At its MTD of 100 mg/kg (qd×5), i.p. administration of HAR 6caused extensive tumor shrinkage in 9 mice (mean shrinkage=94%); thetenth animal died of toxicity. The lower dose of 50 mg/kg caused a mean81% tumor shrinkage in 8 mice, and 82% tumor growth inhibition in theremaining 2 animals. Topotecan at its MTD of 4 mg/kg (i.p.; daily×5)caused a mean 94% tumor shrinkage in all 10 mice.

The approximately 19% body weight loss incurred by mice treated with 100mg/kg HAR 6 (and one drug-related death) establishes that dose as anacceptable MTD for the agent in nude mice according to NCI standards.Topotecan (TPT) at 4 mg/kg was also given at its MTD as evidenced by the15% weight loss observed in the treated mice (no mortality).

The remaining three compounds, HAR 4, 5 and 7, were evaluated againstMX-1 in a separate experiment, including an internal topotecan control.HAR 4 and HAR 7 demonstrated almost equivalent, outstanding activityagainst the MX-1 breast carcinoma xenograft similar to that determinedfor HAR 6. The 20 mg/kg of HAR 4 was toxic, and 10 mg/kg proved to bethe MTD. At the dose of 10 mg/kg (daily×5), HAR 4 caused a mean 77%tumor shrinkage in 8 mice, and the complete disappearance of tumors in 2animals. HAR 7 at the lower dose of 20 mg/kg (40 mg/kg producedlethality) caused a mean 98% tumor shrinkage in all 10 mice. Since 20mg/kg caused virtually no weight loss (or mobility), it is possible thatthe somewhat higher dose of 25 or even 30 mg/kg could be the actual MTDfor HAR 7.

HAR 5 at its solubility limiting dose of 200 mg/kg (weight gained bymice at this dose) caused a mean 73% tumor shrinkage in 3 mice and amean 82% growth inhibition in 7 animals. Topotecan at its MTD of 4 mg/kgproduced one complete regression, six partial responders (mean 94% tumorshrinkage) and two mice with an average 94% tumor growth inhibition.There was one drug-related death in this topotecan group. The topotecanresults in the two MX-1 experiments are therefore quite reproducible.

4) Human Lung And Human Prostate Tumor Models

For the human lung and prostate tumor xenograft models the followingprocedure was used. Nude mice were implanted s.c. by trocar withfragments of human lung or prostate carcinomas harvested from s.c.growing tumors in nude mice hosts. When tumors were approximately 5 mm×5mm in size (usually ten to fourteen days after inoculation), the animalswere pair-matched into treatment and control groups. Each groupcontained 10 tumored mice, each of which was ear-tagged and followedindividually throughout the experiment. The administration of drugs orvehicle begins the day the animals are pair-matched (day 1). The doses,route of drug administration and schedule were selected as appropriatefor the study in question. (See above protocol). If the MTD dose of anagent was not known, it was determined in an initial dosing experimentin non-tumored mice. HAR 7 was given at its MTD and 1/2 MTD doses i.p.on a daily×5 and a daily×1 schedule. Topotecan was run as a positivecontrol at its MTD dose on a daily×5 and a daily×1 schedule. Male nudemice were used for both prostate studies.

The experiment is usually terminated when control tumors reach a size of1-2 g. Mice are weighed twice weekly, and tumor measurements were takenby calipers twice weekly, starting on day 1. These tumor measurementsare converted to mg tumor weight by a well-known formula, and from thesecalculated tumor weights the termination date can be determined. Upontermination, all mice are weighed, sacrificed, and their tumors excised.Tumors are weighed, and the mean tumor weight per group is calculated.In these models, the mean treated tumor weight/mean control tumorweight×100% (T/C) is subtracted from 100% to give the tumor growthinhibition (TGI) for each group.

Some drugs cause tumor shrinkage in the human tumor xenograft models.With these agents, the final weight of a given tumor is subtracted fromits own weight at the start of treatment on day 1. The differencedivided by the initial tumor weight is the % shrinkage. A mean % tumorshrinkage can be calculated from the data from the mice in a group thatexperienced tumor regressions. If the tumor completely disappears in amouse, this is considered a complete regression or complete tumorshrinkage. If desired, mice with partial or total tumor regressions canbe kept alive past the termination date to see whether they live tobecome term, tumor-free survivors.

The results for the human lung and human prostate xenograft models areshown below in Table 10:

                  TABLE 10                                                        ______________________________________                                        Model Compound  Dose         Deaths                                                                              TGI.sup.1                                                                           Shrink.sup.2                         ______________________________________                                        Lung  HAR7      30 mg/kg × 5 days                                                                    0/10  8/10  2/10                                                                    67%   100%                                       TPT        4 mg/kg × 5 days                                                                    0/10  8/10  2/10                                                                    66%   55%                                        HAR7      200 mg single ds                                                                           1/10  7/10  2/10                                                                    64%   67%                                        TPT        8 mg single ds                                                                            0/10  0/10  0/10                                 Prostate                                                                            HAR7      30 mg/kg × 5 days                                                                    0/10  10/10 0/10                                                                    77%                                              TPT        3 mg/kg × 5 days                                                                    0/10  10/10 0/10                                                                    35%                                              HAR7      200 mg single ds                                                                           0/10  10/10 0/10                                                                    80%                                              HAR7      100 mg single ds                                                                           0/10  9/10  1/10                                                                    51%   20%                                        TPT        8 mg single ds                                                                            0/10  0/10  0/10                                 ______________________________________                                         Key:                                                                          1) TGI = Number of Animals Experiencing Tumor Growth Inhibition; and %        Tumor Growth Inhibition Rate.                                                 2) Shrink = Number of Animals Experiencing Tumor Shrinkage; and % Tumor       Shrinkage Rate.                                                          

The compound, HAR 7, was evaluated against both the lung and prostatemodels with an internal topotecan control. HAR7 demonstrated outstandingactivity against the lung and prostate xenograft models. At the dose of30 mg/kg (daily×5), HAR 7 caused the complete disappearance of tumors in2 animals with lung xenografts. At the dose of 30 mg/kg (daily×5), HAR 7caused a mean tumor growth inhibition of 77% in all ten animals havingthe prostate xenograft.

Even more encouraging were the single dose results for HAR7 whichresulted in significant tumor growth inhibition and even some animalsexperiencing partial cures for both the lung and prostate xenograftmodels. By comparison a single dose of TPT was completely ineffectivefor both models. This is indeed a rare occurrence, as single doses ofchemotherapeutic agents are usually ineffective.

EXAMPLE 3 Synthesis Of Novel Camptothecin Analogs

A) Synthesis Of 7-Hydroxymethylcamptothecin

The procedure used to synthesize 7-hydroxymethyl-camptothecin (FIG. 3)was a modified version of the original procedure described by Sawada.See Sawada et al., Chem. Pharm. Bull. 39:2574 (1991)!.

Camptothecin (20.0 g) was dissolved in mixture of methanol (750 mL),water (500 mL), conc. H₂ SO₄ (500 mL) and finely ground FeSO₄ (16.0 g)and hydrogen peroxide (100 mL 30% solution) was added dropwise over thecourse of 2 h and the reaction mixture was allowed to stir for anotherhour. The reaction mixture was then filtered through a sintered glassfunnel to remove all undissolved material. The resultant filtrate wasthen cooled to 0° C. and NaOH (375 g) in 1 L water was added slowly withvigorous stirring. The yellow-brown solid was then filtered, washed withwater and pumped to dryness. The resultant crude product was then placedin a 1 L erlenmyer flask, DMF (300 mL) added and warmed to 110° C. Oncethe solid is dispersed into small particles, acetonitrile (500 mL) wasadded and the mixture heated until boiling. The mixture was then allowedto cool to room temperature then 0° C., filtered and the solid washedwith chloroform to provide 7-hydroxymethylcamptothecin (21.50 g, 99%) asa pale yellow solid.

B) Synthesis Of Tri-O-acetyl-D-glucal

Tri-O-acetyl-D-Glucal was synthesized according to the followingprocedure. Alternatively it could be commercially obtained fromPfanstiehl Laboratories Inc. (Wankeyan, Ill.), however the proceduredescribed below has the advantage of reduced cost compared to thecommercial source.

Glucose (1.000 g) was suspended in a solution of acetic acid (10 mL) andacetic anhydride (3.606 g, 7.0 equiv) and 1,000 g 31% HBr/acetic acidsolution added. The reaction mixture was allowed to stir for 1 h, afterwhich 9.000 g more 31% HBr/acetic acid solution (total of 7.7 equiv HBr)was added and allowed to stir overnight. Sodium acetate was then added(2.700 g) to neutralized the excess HBr, and the reaction mixture wasadded to a suspension containing pulverized CuSO₄. 5H₂ O (0.315 g), zinc(12.600 g), water (10 mL), sodium acetate (9.450 g), and acetic acid (5mL) and the resultant reaction mixture was stirred vigorously for 1.5 h.The solution was then filtered and the solid washed with ethyl acetate(100 mL) and water (100 mL). The organic layer of the filtrate was thenwashed with NaHCO₃ (100 mL) and brine (50 mL), dried (Na₂ SO₄), filteredand the solvent removed under reduced pressure to providetri-O-acetyl-D-glucal (1.350 g, 98%) as a colorless oil free ofimpurities as judged by ¹ H NMR.

C) Synthesis Of Di-O-acetyl-(o-methoxy)benzoyl Glucal

Tri-O-acetyl-D-glucal (1.000 g) was dissolved with o-anisic acid (0,671g, 1.2 equiv.) and iodine (0.186 g, 0.2 equiv.) in 45 mL THF and quicklycooled to -78° C. A 1 mm Hg vacuum line was then attached and thereaction mixture allowed to warm slowly to -5° C. This reaction mixturewas allowed to stir for 2 h under these conditions, replacing the lostTHF solvent periodically. The reaction mixture was then poured into 50mL ethyl acetate and washed successively with saturated Na₂ SO₂ O₃,saturated NaHCO₃, and brine. The organic layer was then dried over Na₂SO₄, filtered and the solvent removed under reduced pressure. Theresultant crude oil was purified by silica gel chromatography (75% ethylacetate in hexane) to provide diacetyl-(o-methoxy)benzoyl glucal (1.141g, 85%) as a mixture of 4 isomers. A 6:1 mixture of the α and β isomerscould be separated from a 1.4:1 mixture of 3R and 3S isomers forspectral analysis by silica gel chromatography (20% ethyl acetate).While isomers are produced using this procedure, under the conditions inwhich the isomeric mixture is subsequently added to the7-hydroxymethylcamptothecin (see below), a single intermediate is formedresulting in a single final stereochemical product.

α anomer: TLC R_(f) 0.56 (2:1 ethyl acetate:hexanes); α!_(D) ²⁰ +18.9°(c=1.02, CHCl₃) ¹ H NMR (360 MHz, CDCl₃) δ2.066 (s, 3H), 2.117 (s, 3H),3.910 (s, 3H), 4.260 (m, 3H), 5.431 (ddd, J=9.5, 3.2, 1.6 Hz, 1H), 5.999(1H) and 6.060 (1H) (ABq, J_(AB) =10.2 Hz, the 5.999 peaks are furthersplit into dd with J=2.8, 1.9 Hz, the 6.060 peaks are further split intodd with J=0.8, 0.8 Hz), 6.563 (ddd, J=2.8, 0.9, 0.9 Hz, 1H), 6.994 (m,2H), 7.505 (ddd, J=8.1, 7.5, 1.8 Hz, 1H), 7.816 (dd, J=8.1, 1.8 Hz, 1H);¹³ C NMR (90 MHz, CDCl₃) δ20.69 (q), 20.91 (q), 55.94 (q), 62.57 (t),64.80 (d), 69.17 (d), 88.33 (d), 112.11 (d), 119.31 (s), 120.09 (d),126.18 (d), 130.53 (d), 131.74 (d), 159.51 (s), 164.56 (s), 170.08 (s),170.77 (s); IR (KBr) 759 (w), 926 (m), 1044 (m), 1193 (w), 1236 (s),1294 (w), 1371 (w), 1438 (w), 1466 (w), 1492 (w), 1601 (w), 1743 (s)cm⁻¹.

3R and 3S isomers: TLC R_(f) 0.62 (2:1 ethyl acetate:hexanes); α!_(D) ²⁰+30.4° (c=1.01, CHCl₃); ¹ H NMR (300 MHz, CDCl₃) δ2.034 (s, 3H, 3Sisomer), 2.0931 (s, 3H, 3R isomer), 2.096 (s, 3H, 3R isomer), 2.099 (s,3H, 3S isomer), 3.900 (s, 3H), 4.218-4.534 (m, 3H), 5.037 (m, 1H), 5.236(dd, J=10.1, 3.7 Hz, 1H, 3R isomer), 5.390 (dd, J=6.8, 5.8 Hz, 1H, 3Sisomer), 5.544 (dd, J=4.6, 3.8 Hz, 1H, 3S isomer), 5.709 (dd, J=6.8, 3.8Hz, 1H, 3R isomer), 6.505 (d, J=6.2 Hz, 1H, 3S isomer), 6.580 (d, J=5.9Hz, 1H, 3R isomer), 7.004 (m, 2H), 7.485 (m, 1H), 7.784 (m, 1H); ¹³ CNMR (90 MHz, CDCl₃) δ20.74 (q, 2C), 55.95 (q), 61.64 (t, 3S isomer),62.04 (t, 3R isomer), 66.67 (d), 67.26 (d, 3R isomer), 67.34 (d, 3Sisomer), 70.78 (d, 3R isomer), 74.04 (d, 3S isomer), 97.77 (d, 3Risomer), 99.16 (d, 3S isomer), 111.98 (d, 3S isomer), 112.15 (d, 3Risomer), 119.90 (s), 120.16 (d), 131.47 (d, 3R isomer), 131.99 (d, 3Sisomer), 133.73 (d, 3R isomer), 134.05 (d, 3S isomer), 145.56 (d, 3Sisomer), 147.92 (d, 3R isomer), 159.30 (s), 165.36 (s), 169.54 (s),170.69 (s); IR (KBr) 758 (w), 1076 (m), 1128 (s), 1227 (w), 1295 (w),1369 (w), 1438, 1468 (w), 1492 (w), 1601 (w), 1647 (w), 1744 (s) cm⁻¹.

D) Synthesis Of 7- 4,6-Di-O-acetyl-2,3dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin (HAR4)

Toluene sulfonic acid monohydrate (4.500 g) was placed in a 100 mLrounded bottomed flask and heated at reduced pressure until all of thesolid was melted and the water removed. THF (30 mL), 7-hydroxymethylcamptothecin (1.500 g) and iodine (0.500 g) was added anddiacetyl-(o-methoxy)benzoyl glucal (5.000 g) in THF (30 mL) was addeddropwise over the course of 2 h and the resultant reaction mixture wasallowed to stir overnight. The reaction mixture was then poured intoethyl acetate (200 mL) and the organic layer was washed with saturatedNa₂ S₂ O₃ (100 mL), saturated NaHCO₃ (100 mL), and brine (50 mL). Theorganic layer was then dried over Na₂ SO₄, filtered, and concentratedand the resultant crude solid purified by silica gel chromatography(gradient of 50% ethyl acetate in hexanes to 100% ethyl acetate) to give7- 4,6-di-O-acetyl-2,3dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin (2.021g, 86%) as a yellow solid.

mp 99°-101° C.; α!_(D) ²⁰ +49.0° (c=1.00, CHCl₃); ¹ H NMR (300 MHz,CDCl₃) δ1.042 (dd, J=7.3, 7.3 Hz, 3H), 1.905 (dq, J=7.3, 7.3 Hz, 2H),2.098 (s, 6H), 3.870 (s, 1H, OH), 4.091-4.279 (m, 3H), 5.187-5.576 (m,7H), 5.739 (d,J=16.4 Hz, 1H), 5.942 (1H) and 6.010 (1H) (ABq, J_(AB)=10.3 Hz, the 5.942 peaks are further split into dd with J=2.0, 2.0 Hz),7.666 (s, 1H), 7.678 (m, 2H), 7.824 (ddd, J=8.3, 6.8, 1.3 Hz, 1H), 8.066(d, J=7.7 Hz, 1H), 8.245 (d, J=8.3 Hz, 1H); ¹³ C NMR (90 MHz, CDCl₃)δ7.63 (q), 20.61 (q), 20.76 (q), 31.45 (t), 50.24 (t), 62.63 (t), 64.89(t), 65.08 (t), 66.11 (d), 67.43 (d), 72.56 (s), 94.37 (d), 97.72 (d),118.54 (s), 123.02 (d), 125.62 (d), 126.38 (d), 127.04 (s), 127.97 (d),130.08 (d), 130.24 (d), 130.41 (d), 138.13 (s) 146.02 (s), 148.71 (s),149.86 (s), 152.34 (s), 157.34 (s), 170.04 (s), 170.48 (s), 173.61 (s);IR (KBr) 1051 (m), 1156 (w), 1231 (s), 1371 (m), 1448 (w), 1512 (w),1610 (m), 1661 (m), 1745 (s), 3459 (w) cm⁻¹.

E) Synthesis Of 7- 2,3-Dideoxy-α-D-erythro-hex-2-enopyranosylcamptothecin (HAR7)

7- 4,6-di-O-acetyl-2,3dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethyl-camptothecin(1.000 g) was placed in a 50 mL rounded bottom flask and 25 mL methanolwas added. The mixture was then cooled to 0° C. and NH₃ was bubbledthrough (8.100 g dissolved) and the reaction mixture was allowed to stirovernight. The solvent was then removed under reduced pressure and theresultant crude purified by silica gel chromatography (5% methanol inethyl acetate) to provide 7-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(0.630 g, 73%) as a light yellow solid.

mp 188° C. (dec.); α!_(D) ²⁰ +13.7° (c=0.40, DMSO); ¹ H NMR (360 MHz,DMSO) δ0.890 (dd, J=7.4, 7.4 Hz, 3H), 1.879 (m, 2 H), 3.434-3.560 (m,3H), 3.874 (m, 1H), 4.692 (dd, J=5.7, 5.7 Hz, 1H, OH), 5.147 (d, J=6.6Hz, 1H), 5.255 (m, 2H), 5.384 (s, 2H), 5.395 (m, 1H), 5.436 (s, 2H),5.745 (1H) and 5.910 (1H) (ABq, J_(AB) =10.0 Hz, the 5.745 peaks arefurther split into dd with J=2.3, 2.3 HZ), 6.542 (s, 1H, OH), 7.349 (s,1H), 7.746 (dd, J=7.1, 7.0 Hz, 1H), 7.880 (ddd, J=8.4, 7.0, 1.3 Hz, 1H),8.194 (dd, J=7.1, 1.3 Hz, 1H), 8.311 (d, J=8.4 Hz, 1H); ¹³ C NMR (90MHz, DMSO) δ172.39 (s), 156.68 (s), 152.36 (s), 149.90 (s), 148.12 (s),145.40 (s), 138.94 (s), 135.22 (d), 130.03 (d), 129.56 (d), 128.31 (s),127.66 (d), 126.02 (s), 124.69 (d), 124.45 (d), 118.99 (s), 96.56 (d),93.72 (d), 73.36 (d), 72.30 (s), 65.18 (t), 63.88 (t), 62.24 (d), 60.65(t), 50.12 (t), 30.21 (t), 7.71 (q); IR (KBr) 1026 (s), 1055 (s), 1159(m), 1231 (w), 1384 (m), 1512 (w), 1597 (s), 1658 (s), 1746 (s), 3402(m) cm⁻¹.

F) Synthesis Of Hexa-O-acetyl maltal

The procedure used to synthesize hexa-O-acetyl maltal (not commerciallyavailable) is described below. This procedure has the advantage of usingthe same solvent for the entire workup. Maltose monohydrate (1.000 g 90%maltose, 10% glucose and maltatriose) was suspended in a solution ofacetic acid (10 mL) and acetic anhydride (2.833 g, 10.0 equiv) and 1.000g 31% HBr/acetic acid solution added. The reaction mixture was allowedto stir for 1 h, after which 9.000 g more 31% HBr/acetic acid solutionwas added and allowed to stir overnight. The reaction mixture was thenpoured into a suspension containing pulverized CuSO₄.5H₂ O (0.182 g),zinc (7.290 g), water (10 mL), sodium acetate (5.470 g), and acetic acid(5 mL) and the resultant reaction mixture was stirred vigorously for 1.5h. The solution was then filtered and the solid washed with ethylacetate (100 mL) and water (100 mL). The organic layer of the filtratewas then washed with NaHCO₃ (100 mL) and brine (50 mL), dried (Na₂ SO₄),filtered and the solvent removed under reduced pressure to provide acolorless oil which was purified by silica gel chromatography (50% ethylacetate/hexanes) to give hexa-O-acetyl-maltal (1.210 g, 86%) as acolorless solid and tri-O-acetyl-D-glucal (0.132 g, 88%) as a colorlessoil. Regarding the maltose starting material, a more pure commercialsample would be preferred, obviating the need for the aforementionedchromatographic separation.

G) Synthesis Of Penta-O-Acetyl-O-(o-methoxy)benzoylmaltal

Hexa-O-acetylmaltal (1.000 g) was dissolved with o-anisic acid (0.326 g,1.2 equiv.) and iodine (0.090 g, 0.2 equiv.) in 45 mL THF and quicklycooled to -78° C. A 1 mm Hg vacuum line was then attached and thereaction mixture allowed to warm slowly to -5° C. This reaction mixturewas allowed to stir for 3 h under these conditions, replacing the lostTHF solvent periodically. The reaction mixture was then poured into 50mL ethyl acetate and washed successively with saturated Na₂ S₂ O₃,saturated NaHCO₃, and brine. The organic layer was then dried over Na₂SO₄, filtered and the solvent removed under reduced pressure. Theresultant crude oil was purified by silica gel chromatography (75% ethylacetate in hexane) to provide pentaacetyl-(o-methoxy)benzoyl maltal(1.103 g, 95%) as a mixture of 4 isomers. An 8:1 mixture of the α and βisomers could be separated from a mixture of 3R and 3S isomers alongwith a small amount of starting hexa-O-acetylmaltal for spectralanalysis by silica gel chromatography (20% ethyl acetate).

α anomer: mp 59°-60° C.; TLC R_(f) 0.48 (2:1 ethyl acetate:hexanes);α!_(D) ²⁰ +118.4° (c=1.03, CHCl₃); ¹ H NMR (360 MHz, CDCl₃) δ2.013 (s,3H), 2.031 (s, 3H), 2.040 (s, 3H), 2.089 (s, 3H), 2.105 (s, 3H),3.373-4.517 (m, 7H), 3.928 (s, 3H), 4.717-5.512 (m, 4H), 5.991 (1H) and6.012 (1H) (ABq, J_(AB) =10.3), 6.526 (br s), 7.014 (m, 2H), 7.521 (dd,J=8.2, 7.5 Hz, 1H), 7.844 (d, J=7.6 Hz, 1H); ¹³ C NMR (90 MHz, CDCl₃)δ20.54 (q, 5C), 55.98 (q), 61.73 (t), 63.05 (t), 68.35 (d, 3C), 69.78(d, 3C), 70.74 (d), 88.21 (d), 94.44 (d), 112.19 (d), 119.53 (s), 120.10(d), 126.24 (d), 129.84 (d), 131.65 (d), 133.83 (d), 159.39 (s), 164.46(s), 169.19 (s), 169.71 (s), 169.82 (s), 170.16 (s, 2C); IR (KBr) cm⁻¹.

H) Synthesis Of 7-6-O-Acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-a-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin(HAR5)

Toluene sulfonic acid monohydrate (0.590 g) was placed in a 25 mLrounded bottomed flask and heated at reduced pressure until all of thesolid was melted and the water removed. THF (10 mL), 7-hydroxymethylcamptothecin (0.200 g) and iodine (0.100 g) was added andpentaacetylbenzoyl maltal (1.000 g) in THF (10 mL) was added dropwiseover the course of 1 h and the resultant reaction mixture was allowed tostir overnight. The reaction mixture was then poured into ethyl acetate(100 mL) and the organic layer was washed with saturated Na₂ S₂ O₃ (50mL), saturated NaHCO₃ (50 mL), and brine (25 mL). The organic layer wasthen dried over Na₂ SO₄, filtered, and concentrated and the resultantcrude solid purified by silica gel chromatography (gradient of 50% ethylacetate in hexanes to 100% ethyl acetate) to give 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(0.280 g, 61%) as a pale yellow solid.

mp 139°-142° C.; α!_(D) ²⁰ +103.1° (c=1.00, CHCl₃); ¹ H NMR (300 MHz,CDCl₃) δ1.026 (dd, J=7.4, 7.4 Hz, 3H), 1.909 (m, 2H), 2.003 (s, 3H),2.025 (s, 3H), 2.060 (s, 3H), 2.102 (s, 3H), 2.155 (s, 3H),4.020-4.4.357 (m, 7H) 4.839 (dd, J=3.9, 10.3 Hz, 1H), 5.065 (dd, J=9.7,9.8 Hz, 1H), 5.165-5.494 (m, 7H), 5.709 (d, J=6.3 Hz, 1H), 5.941 (br s,2H), 7.670 (s, 1H), 7.678 (dd, J=7.5, 7.5 Hz, 1H), 7.817 (dd, J=7.5, 7.5Hz), 8.103 (d, J=7.5 Hz), 8.245 (d, J=7.5 Hz, 1H); ¹³ C NMR (90 MHz,CDCl₃) δ7.83 (q), 20.60 (q), 20.64 (q), 20.69 (q, 2C), 20.87 (q), 31.65(t), 50.17 (t), 63.10 (t), 64.77 (t), 66.28 (t), 67.99 (t), 68.14 (d),68.25 (d), 69.52 (d), 69.72 (d), 70.67 (d), 72.79 (s), 94.10 (d), 94.32(d), 97.97 (d), 118.80 (s), 123.45 (d), 126.13 (d), 126.78 (d), 127.68(s), 128.24 (d), 129.62 (d), 130.31 (d), 130.57 (d), 138.05 (s), 146.25(s), 149.05 (s), 150.03 (s), 152.47 (s), 157.49 (s), 169.54 (s), 169.98(s), 170.34 (s), 170.54 (s), 170.58 (s, 2C), 173.77 (s); IR (KBr) 1038(s), 1137 (w), 1229 (s), 1369 (m), 1436 (w), 1614 (m), 1662 (m), 1748(s), 3471 (w) cm⁻¹.

I) Synthesis Of 7-4-O-(α-D-Glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin(HAR6)

7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(2.600 g) was placed in a 50 mL rounded bottom flask and 25 mL methanolwas added. The mixture was then cooled to 0° C. and NH₃ was bubbledthrough (5.000 g dissolved) and the reaction mixture was allowed to stirovernight. The solvent was then removed under reduced pressure and theresultant crude purified by silica gel chromatography (5% methanol inethyl acetate) to provide 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(1.580 g, 80%) as a light yellow solid.

mp 197°-199° C.; α!_(D) ²⁰ +75.4° (c=1.00, DMF); ¹ H NMR (300 MHz, DMSO)δ0.887 (dd, J=7.3, 7.3 Hz, 3H), 1.873 (m, 2H), 3.037-3.675 (m,), 4.107(d, J=10.2 Hz, 1H), 4.477 (m, 1 H, OH), 4.741 (m, 3H, OH), 4.858 (br s,1H, OH), 4.889 (d, J=4 Hz, 1H), 5.270 and 5.394 (ABq, J_(AB) =12.8 Hz;the 5.394 pm peaks also form an AB pattern with the peaks at 5.440 pm)and 5.440 (ABq, J_(AB) =12.4 Hz), 5.832 (br d, J=10.0 Hz, 1H), 6.160 (d,J=10.0 Hz, 1H), 6.517 (s, 1H, OH), 7.350 (s, 1H), 7.750 (dd, J=7.4, 7.6Hz, 1H), 7.879 (dd, J=7.4, 7.6 Hz, 1H), 8.184 (d, J=7.4 Hz), 8.315 (d,J=7.4 Hz, 1H); ¹³ C NMR (75 MHz, DMSO) δ7.71 (q), 30.24 (t), 50.08 (t),60.72 (t, 2C), 64.01 (t), 65.18 (t), 67.16 (d), 70.00 (d), 70.98 (d),71.43 (d), 72.30 (s), 72.93 (d), 73.39 (d), 93.80 (d), 96.24 (d), 96.58(d), 118.99 (s), 124.41 (d), 125.82 (d), 125.97 (s), 127.68 (d), 128.29(s), 129.53 (d), 130.03 (d), 130.77 (d), 138.76 (s), 145.36 (s), 148.09(s), 149.88 (s), 152.31 (s), 156.66 (s), 172.39 (s); IR (KBr) 1024 (s),1051 (s), 1152 (m), 1231 (w), 1255 (w), 1280 (w), 1401 (w), 1511 (w),1597 (s), 1658 (s), 1745 (s), 3394 (m) cm⁻¹.

J) Synthesis Of Hexa-O-acetyl-lactal

Lactose (1.000 g) was suspended in a solution of acetic acid (10 mL) andacetic anhydride (2.680 g, 9.0 equiv) and 1.000 g 31% HBr/acetic acidsolution added. Although the solid lactose was not dissolved after thereaction mixture was allowed to stir for 1 h, 9.000 g more 31%HBr/acetic acid solution was added and allowed to stir overnight. Thereaction mixture was then poured into a suspension containing pulverizedCuSO₄.5H₂ O (0.182 g), zinc (7.290 g), water (10 mL), sodium acetate(5.470 g), and acetic acid (5 mL) and the resultant reaction mixture wasstirred vigorously for 1.5 h. The solution was then filtered and thesolid washed with ethyl acetate (100 mL) and water (100 mL). The organiclayer of the filtrate was then washed with NaHCO₃ (100 mL) and brine (50mL), dried (Na₂ SO₄), filtered and the solvent removed under reducedpressure to provide a colorless solid which was purified by silica gelchromatography (50% ethyl acetate/hexanes) to give hexa-O-acetyl-lactal(1.010 g, 61%).

K) Synthesis Of Penta-O-Acetyl-O-(o-methoxy)benzoyllactal

Hexa-O-acetyllactal (1.000 g) was dissolved with o-anisic acid (0.326 g,1.2 equiv.) and iodine (0.090 g, 0.2 equiv.) in 45 mL THF and quicklycooled to -78° C. A 1 mm Hg vacuum line was then attached and thereaction mixture allowed to warm slowly to -5° C. This reaction mixturewas allowed to stir for 3 h under these conditions, replacing the lostTHF solvent periodically. The reaction mixture was then poured into 50mL ethyl acetate and washed successively with saturated Na₂ S₂ O₃,saturated NaHCO₃, and brine. The organic layer was then dried over Na₂SO₄, filtered and the solvent removed under reduced pressure. Theresultant crude oil was purified by silica gel chromatography (75% ethylacetate in hexane) to provide penta-O-acetyl-O-(o-methoxy)benzoyl lactal(1.003 g, 86%) as a inseparable mixture of 4 isomers.

L) Synthesis Of 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-a-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin

Toluene sulfonic acid monohydrate (2.100 g) was placed in a 50 mLrounded bottomed flask and heated at reduced pressure until all of thesolid was melted and the water removed. THF (10 mL), 7-hydroxymethylcamptothecin (0.700 g) and iodine (0.350 g) was added andpentaacetyl-(o-methoxy)benzoyl-lactal (3.500 g) in THF (10 mL) was addeddropwise over the course of 2 h and the resultant reaction mixture wasallowed to stir overnight. The reaction mixture was then poured intoethyl acetate (100 mL) and the organic layer was washed with saturatedNa₂ S₂ O (50 mL), saturated NaHCO₃ (50 mL), and brine (25 mL). Theorganic layer was then dried over Na₂ SO₄, filtered, and concentratedand the resultant crude solid purified by silica gel chromatography(100% ethyl acetate) to give 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(1.190 g, 73%) as a pale yellow solid.

mp 143°-146° C.; α!_(D) ²⁰ +44.5° (c=1.00, CHCl₃); ¹ H NMR (300 MHz,CDCl₃) δ1.030 (dd, J=7.2, 7.2 Hz, 3H), 1.912 (m, 2H), 1.978 (s, 6H),2.061 (s, 3H), 2.118 (s, 3H), 2.166 (s, 3H), 4.006-4.329 (m, 7H), 4.638(d, J=7.8 Hz, 1H), 5.018-5.498 (m, 8H), 5.740 (d, J=16.2, 1H), 5.882 (brd, J=10.3 Hz, 1H), 6.213 (d, J=10.3 Hz, 1H), 7.649 (dd, J=7.6, 7.6 Hz,1H), 7.667 (s, 1H), 7.803 (dd, J=7.5, 7.5 Hz, 1H), 8.042 (d, J=7.5 Hz,1H), 8.230 (d, J=7.5 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ7.83 (q), 20.50(q, 2C), 20.61 (q, 2C), 20.81 (q), 31.81 (t), 50.43 (t), 61.31 (t),62.87 (t), 65.05 (t), 66.37 (t), 66.99 (d), 68.25 (d), 68.99 (d), 70.74(d), 70.84 (d), 72.76 (s), 73.03 (d), 94.35 (d), 97.84 (d), 101.89 (d),118.75 (s), 123.34 (d), 126.00 (d), 127.33 (s), 128.02 (d), 130.14 (d),130.55 (d), 131.68 (d), 132.58 (d), 138.26 (s), 146.25 (s), 148.99 (s),149.96 (s), 152.54 (s), 157.45 (s), 169.15 (s), 169.82 (s), 170.02 (s,2C), 170.35 (s, 2C), 173.70 (s); IR (KBr) 1053 (s), 1158 (m), 1228 (s),1371 (m), 1438 (m), 1558 (w), 1614 (m), 1662 (m), 1751 (s), 3470 (w)cm⁻¹.

M) Synthesis Of 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin

7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(1.750 g) was placed in a 50 mL rounded bottom flask and 25 mL methanolwas added. The mixture was then cooled to 0° C. and NH₃ was bubbledthrough (5.000 g dissolved) and the reaction mixture was allowed to stirovernight. The solvent was then removed under reduced pressure and theresultant crude purified by silica gel chromatography (50% methanol inethyl acetate) to provide 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-hydroxymethylcamptothecin(0.976 g, 73%) as a light yellow solid.

mp 180°-182° C.; α!_(D) ²⁰ +15.9° (c=1.00, DMF); ¹ H NMR (300 MHz, DMSO)δ0.894 (dd, J=7.3, 7.3 Hz, 3H), 1.881 (m, 2H), 3.259-3.629 (m,); 4.080(d, J=9.3 Hz, 1H, OH), 4.196 (br s, 1H, OH), 4.397 (d, J=4.5 Hz, 1H,OH), 4.571 (dd, J=5.5, 5.5 Hz, 1H, OH), 4.722 (m, 2H, OH), 4.932 (br s,1H, OH), 5.240-5.436 (m,), 5.788 (br d, J=10.5 Hz, 1H), 6.125 (d, J=10.5Hz, 1H), 6.546 (s, 1H, OH), 7.345 (s, 1H), 7.741 (dd, J=7.5, 7.5 Hz,1H), 7.875 (dd, J=7.5, 7.5 Hz, 1H), 8.175 (d, J=7.5 Hz, 1H), 8.273(d,J=7.5 Hz, 1H); ¹³ C NMR (90 MHz, CDCl₃) δ7.69 (q), 30.23 (t), 50.11 (t),60.20 (t), 60.37 (t), 64.07 (t), 65.18 (t), 68.11 (d), 70.49 (d), 71.12(d), 71.56 (d), 72.29 (s), 73.24 (d), 75.13 (d), 93.17 (d), 96.56 (d),104.69 (d), 119.00 (s), 124.41 (d), 125.39 (d), 125.99 (s), 127.71 (d),128.29 (d), 129.57 (d), 130.05 (d), 133.64 (d), 138.86 (s), 145.39 (s),148.10 (s), 149.88 (s), 152.38 (s), 156.68 (s), 172.37 (s); IR (KBr)1054 (s), 1159 (w), 1232 (w), 1387 (w), 1512 (w), 1599 (s), 1658 (s),1746 (m), 3394 (m) cm⁻¹.

N) Synthesis Of 7- 4,6-di-O-acetyl-2,3dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin

7- 4,6-di-O-acetyl-2,3dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin (0.200 g)was added to p-toluenesulfonylhydrazine (0.960) and NaOAc (1.000 g) in 5mL DMF and 3 mL water and warmed to reflux for 4 h. The reaction mixturewas then allowed to cool, the solvent removed under reduced pressure andpurification by silica gel chromatography (5% methanol in CH₂ Cl₂)provided 7- 4, 6-di-O-acetyl-2,3dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin as a lightyellow solid (0.176 g, 88%).

mp 160°-162° C.; α!_(D) ²⁰ +57.1° (c=1.00, CHCl₃); ¹ H NMR (360 MHz,CDCl₃)δ1.043 (dd, J=7.2, 7.2 Hz, 3H), 1.928 (m, 4H), 2.047 (s, 3H),2.063 (m, 2H) 2.141 (s, 3H), 3.860 (s, 1H, OH), 3.983 (m, 1H), 4.171(1H) and 4.236 (1H) (AB q, J_(AB) =11.4 Hz, the 4.236 peaks are furthersplit into d with J=5.2 Hz), 4.795 (m, 1H), 5.052 (s, 1H), 5.180, (d,J=14.1 Hz, 1H), 5.305 (d, J=16.2 Hz, 1H), 5.448 (m, 3H) 5.743 (d, J=14.1Hz, 1H), 7.672 (s, 1H), 7.700 (dd, J=8.4, 7.6 Hz, 1H), 7.825 (dd, J=8.4,7.6 Hz, 1H), 8.039 (d, J=8.4 Hz, 1H), 8.245 (d, J=7.6 Hz, 1H); ¹³ C NMR(90 MHz, CDCl₃) δ₋₋ 8.02 (q), 21.04 (q), 21.23 (q), 24.29 (t), 28.71(t), 31.85 (t), 50.78 (t) 63.37 (t), 64.52 (t), 66.48 (t), 67.72 (d),69.71 (d), 72.95 (s), 96.92 (d), 98.08 (d), 118.95 (s), 123.34 (d),125.97 (s), 127.21 (s), 128.33 (d), 130.46 (d), 130.81 (d), 138.67 (s),146.39 (s), 149.06 (s), 150.32 (s), 152.75 (s), 157.73 (s), 170.13 (s),170.99 (s), 173.99 (s); IR (KBr) 1043 (m), 1155 (w), 1233 (s), 1368 (w),1440 (w), 1457 (w), 1613 (m), 1661 (m), 1744 (s), 3471 (w) cm⁻¹.

O) Synthesis Of 7-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin

7- 4,6-di-O-acetyl-2,3dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin (0.150 g) wasplaced in a 10 mL rounded bottom flash with 7.5 mL methanol and NH₃bubbled through at 0° C. until 1 g added. The reaction mixture was thenwarmed to room temperature and allowed to stir overnight. The solventwas then removed under reduced pressure and the resultant crude solidpurified by silica gel chromatography (5% methanol in ethyl acetate) toprovide 7-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin (0.077 g,60%) as a light yellow solid.

Alternatively, 7-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin (0.023g) was added to p-toluenesulfonylhydrazine (0.100) and NaOAc (0.096 g)in 2 mL DME and 1 mL water and warmed to reflux for 6 h. The reactionmixture was then allowed to cool, the solvent removed under reducedpressure and purification by silica gel chromatography (5% methanol inethyl acetate) also provided 7-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-hydroxymethylcamptothecin(0.019 g, 83%) as a light yellow solid.

mp 235° C. (dec.); α!_(D) ²⁰ +36.3° (c=1.00, DMSO); ¹ H NMR (300 MHz,DMSO) δ0.896 (dd, J=7.0, 7.0 Hz, 3H), 1.686-1.906 (m, 4H), 3.222-3.500(m, 3H), 3.661 (m, 1H), 4.484 (dd, J=6.0, 5.2 Hz, 1H, OH), 4.791 (d,J=4.6 Hz, 1H, OH), 5.187 (1H) and 5.338 (1H) (AB q, J_(AB) =14.1 Hz),5.367 (s, 2H), 5.431 (s, 2H), 6.538 (s, 1H), 7.342 (s, 1H), 7.729 (dd,J=8.2, 7.1 Hz, 1H), 7.871 (dd, J=8.2, 7.1 Hz, 1H), 8.181 (d, J=8.2 Hz,1H), 8.209 (d, J=7.1 Hz, 1H); ¹³ C NMR (90 MHz, DMSO) δ7.71 (q), 20.69(t), 27.28 (t), 28.69 (t), 50.35 (t), 61.05 (t), 63.11 (t), 64.79 (d),65.18 (t), 72.31 (s), 75.17 (d 95.55 (d), 96.50 (δ), 118.99 (s), 124.19(d), 125.71 (s), 127.67 (d), 129.61 (d), 130.05 (d), 139.17 (s), 145.29(s), 147.97 (s), 149.88 (s), 152.46 (s), 156.64 (s), 172.39 (s); IR(KBr) 1050 (s), 1398 (w), 1602 (s), 1659 (s), 1749 (m) 3385 (w) cm⁻¹.

P) Synthesis Of 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin

7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-αD-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin(0.200 g) was added to a solution of p-toluenesulfonyl-hydrazine (0.400)and NaOAc (0.700 g) in 5 mL DME and 3 mL water and warmed to reflux for6 h. The reaction mixture was then allowed to cool, poured into 50 mLethyl acetate and washed successively with 50 mL water, 50 mL NaHCO₃,and 25 mL brine. The resultant organic layer was then dried (Na₂ SO₄),filtered and the solvent removed under reduced pressure. Silica gelcolumn chromatography (ethyl acetate) provided 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.178 g, 89% as a light yellow powder.

mp 138°-140° C.; α!_(D) ²⁰ +101.0° (c=1.00, CHCl₃); ¹ H NMR (360 MHz,CDCl₃) δ1.044 (dd, J=7.3, 7.3 Hz, 3H), 1.258 (dd, J=7.9, 6.7 Hz, 1H),1.775-2.087 (m, 5H), 1.993 (s, 3H), 2.023 (s, 3H), 2.062 (s, 3H), 2.105(s, 3H), 2.198 (s, 3H), 3.676 (m, 1H), 3.836 (s, 1H), 3.966 (m, 2H),4.102 (m, 1H), 4.305 (m, 3H), 4.826 (dd, J=10.4, 3.9 Hz, 1H),4.997-5.488 (m, 9H), 5.719 (m, 1H), 7.680 (s, 1H), 7.701 (dd, J=7.5, 7.0Hz, 1H), 7.847 (dd, J=8.2, 7.0 Hz, 1H), 8.100 (d, J=7.5 Hz, 1H), 8.270(d, J=8.2 Hz, 1H); ¹³ C NMR (90 MHz, CDCl₃) δ8.03 (q), 20.89 (q, 4C),21.12 (q), 23.65 (t), 28.56 (t), 31.83 (t), 50.59 (t), 61.84 (t), 63.87(t), 63.94 (t), 66.54 (t), 68.38 (d, 2C), 69.93 (d, 2C), 70.72 (d),71.89 (d), 72.96 (s), 93.28 (d), 96.36 (d), 98.08 (d), 118.90 (s),123.56 (d), 126.27 (s), 127.54 (s), 128.39 (d), 130.53 (d), 130.86 (d),138.43 (s), 146.53 (s), 149.25 (s), 150.22 (s), 152.79 (s), 157.70 (s),169.73 (s), 170.23 (s), 170.45 (s), 170.80 (s), 170.91 (s), 174.09 (s);IR (KBr) 1038 (m), 1155 (w), 1229 (s), 1368 (w), 1615 (w), 1663 (w),1749 (s), 3458 (w) cm⁻¹.

Q) Synthesis Of 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin

7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.100 g) was dissolved in 10 mL methanol and cooled to 0° C. and NH₃bubbled through until 1.400 g was added. The resultant reaction mixturewas then warmed to room temperature and allowed to stir for 16 h. Thesolvent was then removed under reduced pressure and the resultant crudesolid purified by silica gel chromatography (25% methanol in ethylacetate) to provide 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.042 g, 55%) as a yellow solid. Alternatively, 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.100 g) was added to p-toluenesulfonyl-hydrazine (0.320) and NaOAc(0.390 g) in 5 mL DME and 3 mL water and warmed to reflux for 4 h. Thereaction mixture was then allowed to cool to room temperature, 3 mL morewater added and to solution cooled to 0° C. The precipitate was thenremoved by vacuum filtration, washed with water and CHCl₃, and pumped todryness to again provide 7-4-O-(α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.074 g, 73%) as a light yellow solid.

mp 225°14 228° C.; α!_(D) ²⁰ +89.4° (c=1.00, DMSO); ¹ H NMR (360 MHz,DMSO) δ0.895 (dd, J=7.2, 7.2 Hz, 3H), 1.673 (m, 2H), 1.881 (m, 3H),2.049 (m, 1H), 3.039 (m, 1H), 3.170 (m, 1H), 3.228-3.692 (m, 10H), 4.490(br s, 1H, OH), 4.63 (br s, 1H, OH), 4.624 (br s, 1H, OH), 4.831 (d,J=3.7 Hz, 1H), 4.832 (br s, 1H, OH), 5.034 (s, 1H, OH), 5.202 (d, J=14.3Hz, 1H), 5.377 (m, 3H), 5.437 (s, 2H), 6.549 (s, 1H, OH), 7.347 (s, 1H),7.744 (dd, J=8.3, 7.0 Hz, 1H), 7.881 (dd, J=7.2, 7.0 Hz, 1H), 8.195 (d,J=7.2 Hz, 1H), 8.228 (d, J=8.3 Hz, 1H); ¹³ C NMR (90 MHz, DMSO) δ7.71(q), 22.47 (t), 28.11 (t), 30.24 (t), 50.35 (t), 60.79 (t), 60.99 (t),63.07 (t), 65.19 (t), 68.53 (d), 70.09 (d), 71.48 (d), 72.31 (s), 72.93(d), 73.09 (d), 73.22 (d), 94.46 (d), 95.54 (d), 96.57 (d), 119.03 (s),124.21 (d), 125.79 (s), 127.72 (d), 127.98 (s), 129.65 (d), 130.08 (d),138.99 (s), 145.31 (s), 148.03 (s), 149.91 (s), 152.50 (s), 156.66 (s),172.39 (s); IR (KBr) 1052 (s), 1602 (m), 1659 (m), 1746 (m), 3421 (s)cm⁻¹.

R) Synthesis Of 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin

7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin(0.200 g) was added to a solution of p-toluenesulfonyl-hydrazine (0.400)and NaOAc (0.700 g) in 5 mL DME and 3 mL water and warmed to reflux for6 h. The reaction mixture was then allowed to cool, poured into 50 mLethyl acetate and washed successively with 50 mL water, 50 mL NaHCO₃,and 25 mL brine. The resultant organic layer was then dried (Na₂ SO₄),filtered and the solvent removed under reduced pressure. Silica gelcolumn chromatography (ethyl acetate) provided 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.175 g, 87% as a light yellow powder.

mp 148°-150° C.; α!_(D) ²⁰ +55.0° (c=1.00, CHCl₃); ¹ H NMR (300 MHz,CDCl₃) δ1.047 (dd, J=7.4, 7.4 Hz, 3H), 1.259 (dd, J=7.9, 6.7 Hz, 1H),1.907 (m, 2H), 1.972 (s, 3H), 2.037 (s, 3H), 2.051 (s, 3H), 2.157 (s,3H), 2.177 (s, 3H), 2.157 (s, 3H), 2.177 (s, 3H), 3.568 (m, 1H), 3.775(s, 1H), 3.895 (m, 2H), 4.085 (m, 2H), 4.162 (m, 1H), 4.302 (br d,J=11.1 Hz, 1H), 4.549 (d, J=7.7 Hz), 4.981 (m, 2H), 5.148 (m, 2H), 5.408(m, 5H), 5.759 (br d, J=6.6 Hz), 7.546 (dd, J=8.4, 6.6 Hz, 1H), 7.678(s, 1H) 7.839 (dd, J=8.4, 6.6 Hz, 1H), 8.077 (d, J=8.4 Hz, 1H), 8.259(d, J=8.4 Hz, 1H); ¹³ C NMR (90 MHz, CDCl₃) δ8.05 (q), 20.86 (q, 4C),21.15 (q), 26.47 (t), 28.96 (t), 31.86 (t), 50.67 (t), 61.38 (t), 63.69(t), 64.07 (t), 66.61 (t), 67.01 (d), 69.02 (d), 70.16 (d 70.79 (d),71.10 (d), 72.96 (s), 76.50 (d), 96.44 (d), 98.09 (d), 102.41 (d),118.92 (s), 123.58 (d), 126.20 (s), 127.46 (s), 128.40 (d), 130.52 (d),130.87 (d), 138.53 (s), 146.54 (s), 149.23 (s), 150.22 (s), 152.81 (s),157.79 (s), 169.73 (s), 170.34 (s), 170.58 (s), 170.97 (s), 174.16 (s);IR (KBr) 1074 (m), 1228 (s), 1369 (w), 1615 (m), 1662 (m), 1751 (s),3473 (w) cm⁻¹.

S) Synthesis Of 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin

7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.100 g) was dissolved in 10 mL methanol, cooled to 0° C., and NH₃bubbled through until 1.200 g were added. The reaction mixture was thewarmed to room temperature and allowed to stir for 16 h. The solvent wasthen removed and the resultant crude solid purified by silica gelchromatography (25% methanol in ethyl acetate) to provide 7-4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin0.027 g, 36%) as a light yellow solid.

Alternatively, 7-6-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.114 g) was added to p-toluenesulfonyl-hydrazine (0.320) and NaOAc(0.390 g) in 5 mL DME and 3 mL water and warmed to reflux for 4 h. Thereaction mixture was then allowed to cool to room temperature, 3 mL morewater added and to solution cooled to 0° C. The precipitate was thenremoved by vacuum filtration, washed with water and CHCl₃, and pumped todryness to again provide 7- 4-O-(β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin(0.072 g, 63%) as a light yellow solid.

mp 214°-217° C.; α!_(D) ²⁰ +37.4° (c=1.00, DMSO); ¹ H NMR (300 MHz,DMSO) δ0.869 (dd, J=7.3, 7.3 Hz, 3H), 1.744 (m, 2H), 1.868 (m, 3H),2.004 (m, 1H), 3.230-3.639 (m, 10H), 4.176 (d, J=5.5 Hz, 1H), 4.402 (brs, 1H, OH), 4.516 (br s, 1H, OH), 4.563 (br s, 1H, OH), 4.710 (m, 1H,OH), 4.926 (m, 1H, OH), 5.008 (s, 1H), 5.225 (1H) and 5.345 (1H) (AB q,J_(AB) =13.7 Hz), 5.403 (s, 2H), 5.440 (s, 2H), 6.550 (s, 1H), OH),7.350 (s, 1H), 7.761 (dd, J=8.1, 6.7 Hz), 7.891 (dd, J=8.4, 6.7 Hz, 1H),8.205 (d, J=8.4 Hz, 1H), 8.247 (d, J=8.1 Hz, 1H); ¹³ C NMR (90 MHz,DMSO) δ7.70 (q), 26.22 (t), 28.62 (t), 30.22 (t), 50.29 (t), 60.30 (t),60.68 (t), 63.26 (t), 65.18 (t), 67.95 (d), 70.67 (d), 72.30 (s), 73.18(d), 73.34 (d), 74.52 (d), 75.07 (d), 95.65 (d), 96.55 (d), 104.53 (d),119.02 (s), 124.19 (d), 125.76 (s), 127.70 (d), 127.91 (s), 129.63 (d),130.07 (d), 138.99 (s), 145.29 (s), 147.99 (s), 149.88 (s), 152.46 (s),156.65 (s), 172.40(s); IR (KBr) 1031 (s), 1606 (s), 1659 (s), 1749 (s),3450 (s) cm⁻¹.

T) Synthesis Of Camptothecin 20-n-Hexanoate

Camptothecin (0.700 g) and n-hexanoyl chloride (2.20 mL) were dissolvedin 110 mL of a 2:1 mixture of DMF and pyridine and the solution washeated at 80° C. for 6 hours. The reaction mixture was then poured into200 mL of CH₂ Cl₂ and the resulting mixture was washed successively withwater (2×200 mL), 200 mL of 5% aqueous HCl, and 100 mL of brine. Theorganic layer was dried over anhydrous Na₂ SO₄ and concentrated underreduced pressure. The crude solid thus obtained was purified by silicagel flash chromatography with ethyl acetate as the eluent to givecamptothecin 20-n-hexanoate (0.502 g, 56%) as a light yellow solid: mp250°-252° C.; α!_(D) ²⁰ -56.0° (c=1.00, CHCl₃); ¹ H NMR (300 MHz, CDCl₃)δ0.854 (diffused t, 3H), J=7.6 Hz), 0.978 (t, 3H, J=7.5 Hz), 1.25-1.40(m, 4H), 1.60-1.72 (m, 2H) 2.17 (1H) and 2.31 (1H) (ABq, J_(AB) =13.8Hz; both the 2.17 and 2.31 peaks are further split into q with J=7.6 and7.7 Hz, respectively), 2.48 (1H) and 2.49 (1H) (ABq, J_(AB) =14.6 Hz;both the 2.48 and 2.49 peaks are further split into t with J=7.3 and 7.6Hz, respectively), 5.29 (s, 2H), 5.42 (1H), 5.68 (1H), (ABq, J_(AB)=14.3 Hz), 7.22 (s, 1H), 7.67 (ddd, 1H, J=8.1, 6.9, 1.2 Hz, 1H), 7.84(ddd, 1H, J=8.5, 6.9, 1.5 Hz), 7.94 (dd, J=8.1, 1.5 Hz, 1H), 8.21 (dd,J=8.5, 1.2 Hz, 1H), 8.40 (s, 1H); ¹³ C NMR (90 MHz, CHCl₃) δ7.75 (q),14.04 (q), 22.48 (t), 24.50 (t), 31.32 (t), 32.04 (t), 33.96 (t), 50.07(t), 67.28 (t), 75.82 (s), 96.18 (d), 120.50 (s), 128.19 (d), 128.35(s), 128.40 (d), 128.64 (s), 129.76 (d), 130.84 (d), 131.36 (d), 146.17(s), 146.37 (s), 149.04 (s), 152.57 (s), 157.54 (s), 167.78 (s),172.98(s); IR (KBr) 722 (w), 756 (w), 1043 (w), 1229 (w), 1404 (m), 1627(m), 1673 (m), 1743 (s), 1758 (s) cm⁻¹.

EXAMPLE 4

Synthesis Of Novel Glycosylated Hexacyclic Camptothecin Analogs

In their 1994 paper, Sugimori et al described the synthesis of novelhexacyclic camptothecin compounds. See Sugimori et al. "AntitumorAgents. Synthesis And Antitumor Activity Of Novel HexacyclicCamptothecin Analogues," J. Med. Chem. 37:3033 (1994)!. Sugimori andcoworkers were able to synthesize the novel hexacyclic camptothecincompounds which have an additional 5-, 6-, or 7-membered ring cyclizedat positions 7 and 9 of camptothecin by intramolecular cyclization ofpentacyclic camptothecin compounds or Friedlander condensation of theappropriate bicyclic amino ketone and tricyclic ketone. All of thehexacyclic camptothecin compounds synthesized had comparable in vitroactivity compared to SN-38 (the active metabolite of CPT-11), and someeven had superior in vivo anti-cancer activity. However, none wereglycosylated by Sugimori and coworkers.

The present invention contemplates preparation of a novel glycosylatedhexacyclic camptothecin analog by reaction of a hexacyclic camptothecincompound with one or more appropriate alkylhydroxyl linkers to form ahexacyclic camptothecin analog possessing free hydroxyl groups, followedby glycosylation as described above to form the novel glycosylatedhexacyclic camptothecin analog. A representative contemplated novelglycosylated hexacyclic camptothecin analog is illustrated in FIG. 20.

From the above, it should be evident that the analogs of the presentinvention provide a powerful anticancer therapeutic agent. In side byside in vivo studies, novel compounds of the present invention werefound to be superior to existing derivatives in both continuous andsingle dose treatment protocols.

We claim:
 1. A glycosylated analog of camptothecin selected from thegroup consisting of 7-4,6-Di-O-acetyl-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!oxymethylcamptothecin, 7-6-O-Acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2enopyranosyl!oxymethylcamptothecin, 7-4-O-(α-D-Glucopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin,7- 2,3-Dideoxy-α-D-erythro-hex-2-enopyranosyl camptothecin, 7-6-O-Acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin,7-4-O-(β-D-Galactopyranosyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranosyl!-oxymethylcamptothecin,7-4,6-Di-O-acetyl-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin,7- 2,3-Dideoxy-α-erythro-hexanopyranosyl!-oxymethylcamptothecin, 7-6-O-Acetyl-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin,7-4-O-(α-D-Glucopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin,7-6-O-Acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3-dideoxy-α-D-erythro-hexanopyranosyl!-oxymethylcamptothecin,and 7-4-O-(β-D-Galactopyranosyl)-2,3-D-erythro-hexanopyranosyl-oxymethylcamptothecin.2. A pharmaceutical composition comprising the glycosylated analog ofclaim 1, wherein said analog is in an aqueous solution.
 3. Thecomposition of claim 2, wherein said aqueous solution comprisesphysiological saline.
 4. The composition of claim 2, wherein saidaqueous solution comprises an additive selected from the groupconsisting of dextrose, glycerol, mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose and magnesium carbonate.